Methods relating to circulating tumor cell clusters and the treatment of cancer

ABSTRACT

Described herein are methods and assays relating to the presence and/or level of circulating tumor cells (CTCs). These CTC-Cs represent a highly metastatic subpopulation of CTCs. In some embodiments, the methods and assays described herein relate to the treatment of cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation under 35 U.S.C. § 120 of co-pendingU.S. application Ser. No. 16/037,512 filed Jul. 17, 2018, which is acontinuation under 35 U.S.C. § 120 of U.S. application Ser. No.15/031,048 filed Apr. 21, 2016 now U.S. Pat. No. 10,053,692 issued Aug.21, 2018, which is a 35 U.S.C. § 371 National Phase Entry Application ofInternational Application No. PCT/US2014/060610 filed Oct. 15, 2014,which designates the U.S. and claims benefit under 35 U.S.C. § 119(e) ofU.S. Provisional Application No. 61/893,397 filed Oct. 21, 2013,61/908,236 filed Nov. 25, 2013, and 61/918,923 filed Dec. 20, 2013, thecontents of which are incorporated herein by reference in theirentireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 8, 2014, isnamed 030258-079173-PCT_SL.txt and is 11,455 bytes in size.

TECHNICAL FIELD

The technology described herein relates to the diagnosis and treatmentof cancer.

BACKGROUND

The current model of blood-borne metastasis is based on sequential stepsstarting from movement of primary tumor cells into the bloodstream,survival in the circulation, movement of the tumor cells from thebloodstream into a new tissue, and the founding of a new tumor.Circulating tumor cells (CTCs) have been detected in the majority ofepithelial cancers (Yu et al. JCB 2011 192:373) and they hold the key tounderstanding early dissemination events in cancer. Interestingly, thenumber of CTCs largely exceeds the number of metastatic lesions inpatients, indicating that CTCs are not equally capable of causingmetastasis. It is likely that the majority of CTCs die in thebloodstream, with only a minor fraction representing viable metastaticprecursors. The identification of the metastatic pool within CTCs hasthe potential to refine the understanding of cancer metastasis and canpermit the development of new agents for the treatment of metastatichuman tumors.

SUMMARY

As described herein, the inventors have discovered that a subset ofcirculating tumor cells (CTCs) exist as CTC clusters (CTC-Cs). Moreover,the inventors have found that these CTC-Cs represent a highly metastaticsubpopulation of CTCs. Markers of these CTC-Cs and accordingly, methodsof detecting the presence and/or level of CTC-Cs are described herein,as are methods relating to reducing metastasis by inhibiting the growth,survival, and/or metastatic potential of the CTC-Cs.

In one aspect, described herein is an assay comprising measuring thelevel of circulating tumor cell (CTC) clusters in a sample obtained froma subject with a breast or epithelial cancer and determining the subjectto be at increased risk of metastasis of the cancer if the level of CTCclusters is increased relative to a control level. In some embodiments,the level of CTC clusters is measured by measuring the expression levelof a CTC cluster (CTC-C) marker gene in the sample obtained from thesubject; wherein the CTC-C marker gene is a gene selected from the listof FIG. 9. In some embodiments, the level of CTC clusters is measured bymeasuring the expression level of a CTC cluster (CTC-C) marker gene inthe sample obtained from the subject; wherein the CTC-C marker gene is agene selected from the list of Table 2, 3 and/or 4. In some embodiments,the CTC-C marker gene is plakoglobin. In some embodiments, theexpression level of a CTC-C marker gene in circulating tumor cells inthe sample is measured. In some embodiments, the expression level of aCTC-C marker gene in cancer cells obtained from the subject is measured.In some embodiments, the level of CTC clusters is measured using a^(HB)CTC-Chip. In some embodiments, the subject is a subject in need oftreatment for cancer. In some embodiments, an increased level of CTCclusters is a level at least 1.5× greater than the control level. Insome embodiments, an increased level of plakoglobin expression is alevel at least 1.5× greater than the control level.

In one aspect, described herein is a method of determining if a subjectis at increased risk of metastasis, the method comprising measuring thelevel of circulating tumor cell (CTC) clusters in a sample obtained froma subject with a breast or epithelial cancer and determining the subjectto be at increased risk of metastasis of the cancer if the level of CTCclusters is increased relative to a control level. In some embodiments,the level of CTC clusters is measured by measuring the expression levelof a CTC cluster (CTC-C) marker gene in the sample obtained from thesubject; wherein the CTC-C marker gene is a gene selected from the listof FIG. 9. In some embodiments, the level of CTC clusters is measured bymeasuring the expression level of a CTC cluster (CTC-C) marker gene inthe sample obtained from the subject; wherein the CTC-C marker gene is agene selected from the list of Table 2, 3 and/or 4. In some embodiments,the CTC-C marker gene is plakoglobin. In some embodiments, theexpression level of a CTC-C marker gene in circulating tumor cells inthe sample is measured. In some embodiments, the expression level of aCTC-C marker gene in cancer cells obtained from the subject is measured.In some embodiments, the level of CTC clusters is measured using a^(HB)CTC-Chip. In some embodiments, the subject is a subject in need oftreatment for cancer. In some embodiments, an increased level of CTCclusters is a level at least 1.5× greater than the control level. Insome embodiments, an increased level of plakoglobin expression is alevel at least 1.5× greater than the control level.

In one aspect, described herein is a method of reducing the level ofcirculating tumor cell (CTC) clusters in a subject with cancer, themethod comprising reducing the level of expression or activity of aCTC-C marker gene; wherein the CTC-C marker gene is a gene selected fromthe list of FIG. 9. In some embodiments, the CTC-C marker gene is a geneselected from the list of Table 2, 3 and/or 4. In some embodiments,reducing the level of expression or activity of a CTC-C marker genecomprises administering a CTC-C marker gene inhibitory nucleic acid. Insome embodiments, the inhibitory nucleic acid is a siRNA. In someembodiments, the CTC-C marker gene is plakoglobin.

In one aspect, described herein is a method of treating cancermetastasis, the method comprising reducing the level of expression oractivity of a CTC-C marker gene; wherein the CTC-C marker gene is a geneselected from the list of FIG. 9. In some embodiments, the CTC-C markergene is a gene selected from the list of Table 2, 3 and/or 4. In someembodiments, reducing the level of expression or activity of a CTC-Cmarker gene comprises administering a CTC-C marker gene inhibitorynucleic acid. In some embodiments, the inhibitory nucleic acid is asiRNA. In some embodiments, the CTC-C marker gene is plakoglobin.

In one aspect, described herein is a method of treating cancer, themethod comprising measuring the level of circulating tumor cell (CTC)clusters in a sample obtained from a subject with a breast or epithelialcancer; administering a treatment to prevent or reduce metastasis if thelevel of CTC clusters is increased relative to a control level; and notadministering a treatment to prevent or reduce metastasis if the levelof CTC clusters is not increased relative to a control level. In someembodiments, the treatment to prevent or reduce metastasis is selectedfrom the group consisting of a method of treating cancer metastasis asdescribed in the foregoing paragraph; chemotherapy; radiation therapy;or removal of a tumor. In some embodiments, not administering atreatment can comprise a clinical approach of monitoring withouttherapeutic intervention. In some embodiments, the level of CTC clustersis measured by measuring the expression level of a CTC cluster (CTC-C)marker gene in the sample obtained from the subject; wherein the CTC-Cmarker gene is a gene selected from the list of FIG. 9. In someembodiments, the level of CTC clusters is measured by measuring theexpression level of a CTC cluster (CTC-C) marker gene in the sampleobtained from the subject; wherein the CTC-C marker gene is a geneselected from the list of Table 2, 3 and/or 4. In some embodiments, theCTC-C marker gene is plakoglobin. In some embodiments, the expressionlevel of a CTC-C marker gene in circulating tumor cells in the sample ismeasured. In some embodiments, the expression level of a CTC-C markergene in cancer cells obtained from the subject is measured. In someembodiments, the level of CTC clusters is measured using a^(HB)CTC-Chip. In some embodiments, the subject is a subject in need oftreatment for cancer. In some embodiments, an increased level of CTCclusters is a level at least 1.5× greater than the control level. Insome embodiments, an increased level of plakoglobin expression is alevel at least 1.5× greater than the control level.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B demonstrate that the presence of CTC-clusters in breastcancer patients correlates with increased disease progression. FIG. 1Adepicts a graph demonstrating that a total of 79 patients and 265timepoints were analyzed for the presence of single CTCs andCTC-clusters, with 54 of the 79 patients scoring positive for CTCs. Thebar graph shows the percentage of the CTC-positive patients havingsingle CTCs-only (grey), CTC-clusters during one timepoint (white) andCTC-clusters during multiple timepoints (blue). FIG. 1B depicts aKaplan-Meier progression-free survival plot showing progression ratesfor single CTCs-enriched (mean progression-free survival time 160.6days) versus CTC-clusters-enriched (mean progression-free survival time76.1 days) patients. P=0.063 by Log-rank test.

FIGS. 2A-2C demonstrate that CTC-clusters harbor an increased metastaticpotential compared to single CTCs. FIG. 2A depicts a bar graph of themean percentage of cleaved caspase 3-positive cells in lungs fromLM2-SCs- or LM2-CLs-injected mice. n=4, *P<0.02 by Student's t test.

FIG. 2B depicts bar graphs of the mean percentage of single CTCs versusCTC-clusters captured by the ^(HB) CTC-Chip (top) as well as the meanpercentage of single CTCs-versus CTC-clusters-derived lung foci(bottom). n=5. FIG. 2C depicts a bar graph of the normalized metastaticpotential of single CTCs and CTC-clusters. n=5, *P=0.031 by Student's ttest.

FIG. 3 demonstrates that CTC-clusters are oligoclonal units associatedto a faster clearance rate from the bloodstream. Depicted is a graph ofsingle CTCs and CTC-clusters clearance curves. n=5 for single CTCs andn=4 for CTC-clusters, *P<0.01 by two-way ANOVA.

FIGS. 4A-4D demonstrate that RNA sequencing of CTC-clusters and singleCTCs reveals a CTC-clusters-associated gene set. FIG. 4A depicts aschematic of the experiment. Breast cancer patient-derived blood sampleswere processed with the ^(neg)CTC-iChip to obtain a CTCs-enrichedproduct. Live staining was then performed to label CTCs (green) andwhite blood cells (red). Single CTCs and CTC-clusters were isolated witha micromanipulator and processed for RNA sequencing. FIG. 4B depicts aheatmap demonstrating unsupervised hierarchical clustering of 15 singleCTCs pools and matched 14 CTC-clusters isolated from 10 breast cancerpatients. FIG. 4C depicts a heatmap demonstrating the top 31 transcriptsupregulated in CTC-clusters. n=15 for single CTCs and n=14 forCTC-clusters; q<0.01, log 2 fold change >1 in more than 70%intra-patient comparisons. FIG. 4D depicts a graph demonstratingPlakoglobin fold increase in matched CTC-clusters versus single CTCs.The threshold line represents a q<0.01 and log 2 fold increase >1.

FIGS. 5A-5F demonstrate that Plakoglobin is required for CTC-clustersformation and lung metastasis. FIG. 5A depicts a bar graph demonstratingthe relative cell-to-cell adhesion in a panel or mammary epithelialcells and breast cancer cell lines in the presence or absence ofPlakoglobin. n=5: *P<0.04. FIG. 5B depicts LM2 tumor growth curves inthe presence or absence of Plakoglobin. n=4; NS=not significant. FIG. 5Cdepicts bar graphs demonstrating the normalized number of single CTCs(left) and CTC-clusters (right) per ml of blood. Blood samples wereisolated 4 weeks upon primary tumor development and processed with the^(HB)CTC-Chip. n=4; *P<0.05 by Student's t test. FIG. 5D depicts a bargraph demonstrating the normalized lung photon counts from mice bearinga LM2 CTRL or Plakoglobin knockdown primary tumor for 4 weeks. n=4;*P<0.045 by Student's t test. FIG. 5E depicts Kaplan-Meier distantmetastasis-free survival plot demonstrating progression rates forpatients whose primary tumor expressed either “low Plakoglobin” or “highPlakoglobin” transcript. n=1353; P=8.2e^(−0.5) by Log-rank test. FIG. 5Fdepicts a schematic demonstrating that “high Plakoglobin” regions in theprimary tumor are likely to generate CTC-clusters with high metastaticpotential.

FIGS. 6A-6B demonstrate the metastasis potential of LM2-SCs and LM2-CLs.FIG. 6A depicts a graph of lung metastasis growth curve from miceinjected with LM2-SCs or LM2-CLs. n=4; *P<0.03 by Student's t test. FIG.6B depicts a graph of Kaplan-Meier survival plots showing survival ratesfor mice injected with LM2-SCs or LM2-CLs. n=4; P<0.016 by Log-ranktest.

FIG. 7 depicts a heatmap showing expression levels of CTCs-associatedtranscripts (Keratin 7, 8, 18, 19, Cadherinl, EpCAM, EGFR, ErbB2, Metand Cadherinll) and white blood cells-associated transcripts(PTPRC/CD45, CD14 and CD16) in the 15 single CTCs and 14 CTC-clusterssamples used to derive CTC-clusters upregulated transcripts.

FIGS. 8A-8B depict the expression level of Plakoglobin. FIG. 8A depictsimmunoblots demonstrating the expression levels of Plakoglobin andβ-Actin (loading control) in lysates from a panel of non-transformedmammary epithelial cells (HMEC, MCF10A) and breast cancer cell lines(LM2, BT474, MCF7, T47D, BT549, BT20, ZR-75-1) grown in the presence orabsence of Plakoglobin. FIG. 8B depicts an immunoblot demonstrating theexpression levels of Plakoglobin and β-Actin (loading control) inlysates from LM2 xenografts grown in the presence or absence ofPlakoglobin for 30 days.

FIG. 9 depicts a table of upregulated transcripts in CTC-clusters versussingle CTCs. Values represent fold-change.

FIGS. 10A-10D demonstrate that desmosome and adherence junctionspolypeptides are upregulated in CTC-C cells. FIG. 10A depicts a heatmapof expression levels of desmosomes (top) and adherence junctions(bottom) marker genes in the 15 single CTCs and 14 CTC-clusters samplesused to derive CTC-clusters upregulated transcripts. FIG. 10B depicts aheatmap representing fold change of desmosomes (top) and adherencejunctions (bottom) marker genes in all “CTC-clusters vs single CTCs”intrapatient comparisons. FIG. 10C depicts a heatmap of the fold changeof desmosome and adherence junction metagenes in all “CTC-clusters vssingle CTCs” intrapatient comparisons. FIG. 10D is a representation ofthe frequency of “CTC-clusters vs single CTCs” pairs with q<0.01 andfold change >2 for randomly generated metagenes of the same size asdesmosomes (top) and adherence junctions (bottom). Actual number of“CTC-clusters vs single CTCs” pairs with q<0.01 and fold change >2 fordesmosomes (top) and adherence junctions (bottom) metagenes is shown asa line.

FIGS. 11A-11E demonstrate that CTC Clusters Demonstrate IncreasedMetastatic Potential Compared to Single CTCs. FIG. 11A depicts aschematic of the experiment. MDA-MB-231-LM2 (LM2) cells expressing GFP(LM2-GFP) or mCherry (LM2-mCherry) cells were mixed at 1:1 ratio andinjected in the right mammary gland of immunodeficient mice to generateone-color single CTCs and multicolor CTC clusters. Accordingly,one-color metastatic foci are derived from a single CTC, whilemulticolor foci arise predominantly from a CTC cluster. FIG. 11B depictsbar graphs showing the mean percentage of one-color versus multicolorCTC events captured by the HBCTC-Chip (left), the mean percentage ofone-color versus multicolor CTC clusters (middle), as well as the meanpercentage of one-color versus multicolor lung foci (right). n=5. FIG.11C depicts a bar graph showing the normalized metastatic potential ofsingle CTCs and CTC clusters. Error bars represent SEM. n=5, *p=0.031 byStudent's t test. FIG. 11D depicts a schematic of the experiment.LM2-GFP cells were injected in the right mammary gland while LM2-mCherrycells were injected in the left mammary gland of immunodeficient mice togenerate tumors that give rise to one-color single CTCs and CTCclusters, as well as rare multicolor CTC clusters (resulting fromaggregation events). Accordingly, one-color metastatic foci are derivedfrom a single CTC or a CTC cluster, while multicolor foci derive fromCTC aggregates. FIG. 11E depicts bar graphs showing the mean percentageof one-color versus rare multicolor CTC events captured by theHBCTC-Chip (left), the mean percentage of one-color versus multicolorCTC clusters (middle), as well as the mean percentage of one-colorversus multicolor lung foci (right). n=5.

FIGS. 12A-12B demonstrate that CTC Clusters Are More Resistant toApoptosis at Distal Metastatic Sites. FIG. 12A depicts representativebioluminescence images of mice at 0, 6, and 12 days after tail veininjection with LM2-SC or LM2-CL cells (left). n=4. Representative imagesof GFP-stained sections of mouse lungs after injection with LM2-SC orLM2-CL cells (right). FIG. 12 B depicts a bar graph of the meanpercentage of GFP-positive cells in lungs from LM2-SC- orLM2-CL-injected mice. Error bars represent SEM. n=4; NS, notsignificant, *p=0.03 by Student's t test.

FIG. 13 demonstrates that CTC Clusters Demonstrate a Faster ClearanceRate from the Bloodstream. Decpited is a schematic showing theexperimental setup for measuring the clearance time of single CTCs andCTC clusters. Briefly, DiD-stained LM2 cells were prepared as LM2-SC orLM2-CL and injected into the tail vein of immunodeficient mice. In vivoflow cytometry was applied to the ear blood vessels to detect singleCTCs and CTC clusters over a 55 min period after injection. Graphs showrepresentative fluorescence peaks corresponding to the transit of asingle CTC or CTC cluster through the ear blood vessel.

FIGS. 14A-14D demonstrate that the Presence of CTC Clusters in Patientswith Cancer Correlates with Poor Prognosis. FIG. 14A depicts the resultsof a total of 79 breast cancer patients (corresponding to 265 timepoints) analyzed for the presence of CTCs, with 54 of the 79 patientsscoring positive for CTCs. The bar graph shows the percentage ofCTC-positive patients having CTC clusters during more than three timepoints (dark grey), CTC clusters across one to three time points (lightgray) or single CTCs only (black). FIG. 14B depicts kaplan-Meierprogression-free survival plot showing progression rates for breastcancer patients having CTC clusters during more than three time points(dark grey), CTC clusters across one to three time points (light grey)or single CTCs only (black). The mean progression-free survival time foreach group is given in parentheses. p=0.0002 by log rank test. FIG. 14Cdepicts results of a total of 64 prostate cancer patients (correspondingto 202 time points) analyzed for the presence of CTCs, with 48 of the 64patients scoring positive for CTCs. The bar graph shows the percentageof CTC-positive patients having CTC clusters during at least one timepoint or single CTCs only (black). FIG. 14D depicts Kaplan-Meier overallsurvival plot showing progression rates for prostate cancer patientshaving CTC clusters during at least one time point or single CTCs only(black). The mean overall survival time for each group is given inparentheses. p=0.0001 by log rank test.

FIG. 15 demonstrates that Plakoglobin Expression Correlates withDecreased Distant Metastasis-Free Survival. The bar graph of plakoglobinreads per million in matched single CTCs and CTC clusters isolated fromthe same patient. Error bars represent SEM. n=3; *p=0.031.

FIG. 16 demonstrates that Plakoglobin Is Required for CTC ClusterFormation and Lung Metastasis. Lung metastasis growth curves from miceinjected with LM2-GFP-Lucife,ase (left) or BT474-GFP-Lucife,ase (right)cells expressing control or plakoglobin shRNAs and prepared as singlecells (SC) or clusters (CL). Error bars represent SEM. n=4; *p<0.05,**p<0.04 by Student's t test. LM2-GFP-Luciferase tumor growth curves inthe presence or absence of plakoglobin. n=4; NS, not significant.

FIGS. 17A-17D demonstrate that Counts of One-Color and Multicolor Eventsin the LM2 and 4T1 Xenografts. FIG. 17A depicts a table showing countsof one color versus multicolor events within CTCs and lung foci fromboth the “LM2-GFP/LM2-mCherry 1:1” and the “LM2-GFP (right) andLM2-mCherry (left)” models. Results represent means±SEM. FIG. 17Bdepicts a distribution curve describing the expected numbers of GFP- ormCherry-only CTC clusters per mouse given our experimental setup, withthe actual value shown as a red dashed line (top). Blood samples wereisolated 5 weeks after primary tumor development. FIG. 17C depicts atable showing counts of one color versus multicolor events within CTCsand lung foci from both the “4T1-GFP/4T1-mCherry 1:1” and the “4T1-GFP(right) and 4T1-mCherry (left)” models. Mice were sacrificed for CTCsand lungs isolation 3 weeks after primary tumor development. Resultsrepresent means±SEM (n=4) (left). FIG. 17D depicts a bar graph showingthe normalized metastatic potential of 4T1 single CTCs and CTC clusters.n=4, *p<0.036 by Student's t test.

FIGS. 18A-18C demonstrate that BT474 and 4T1 Clusters Are More Resistantto Apoptosis at Distal Metastatic Sites. FIG. 18A depicts bar graphs ofthe mean per-centage of GFP-positive cells in lungs from mice injectedwith BT474 or 4T1 SC versus CL (right). n=4; NS=not significant,*p=0.003 **p=0.002 by Student's t test. FIG. 18b depicts bar graphs ofthe mean percentage of cleaved caspase 3-positive cells in lungs frommice injected with BT474 or 4T1 SC versus CL. n=4; *p=0.037 **p=0.028 byStudent's t test. FIG. 18C depicts lung metastasis growth curves frommice injected with BT474 or 4T1 SC versus CL. n=4; ***p<0.02 *p<0.027*p<0.05 by Student's t test.

FIG. 19 demonstrates Analysis of the Cellular Composition of CTCClusters, Depicted is a heatmap showing expression levels of CTCs-,leukocytes-, T cells-, B cells-, dendritic cells-, natural killer (NK)cells-, hematopoietic stem cells-, macrophages/monocytes-,granulocytes-, platelets-, endothelial cells- and fibroblasts-associatedtranscripts in the 15 single CTCs and 14 CTC clusters samples used toderive CTC clusters upregulated transcripts.

FIG. 20 depicts Kaplan-Meier Plots of CTC-Clusters-Associated Genes.Kaplan-Meier distant metastasis-free survival plots showing progressionrates for patients whose primary tumor expressed either “low” or “high”levels of the top CTC-clusters-associated marker genes.

DETAILED DESCRIPTION

As described herein, the inventors have discovered that clusters ofcirculating tumor cells (CTC-Cs) have a particularly high metastaticpotential. Accordingly, these CTC-Cs are both a diagnostic andtherapeutic target for the management and treatment of cancer. Providedherein are methods of diagnosis, prognosis, and treatment relating toCTC-Cs and their propensity to give rise to metastases.

As used herein, “circulating tumor cells” or “CTCs” refer to tumor cellswhich are shed from a tumor and present in the blood, i.e. incirculation. Cell surface markers that can be used to identify and/orisolate CTCs from other components of the blood are described belowherein. Markers of CTCs, as well as methods of isolating and/ordetecting them are described, e.g. in Yu et al. JBC 2011 192:373; whichis incorporated by reference herein in its entirety.

Some of these CTCs can be present in a cancer patient as CTC-clusters(CTC-Cs). As used herein, “CTC-clusters,” “CTC clusters,” or “CTC-Cs”refer to adherent groups of at least two or more (e.g. 2 or more, 3 ormore, 4 or more, 5 or more, or more) CTCs, i.e. cells that are positivefor one or more cancer cell markers and having intact nuclearmorphology. Cancer cell markers can vary according to the type of cancerand appropriate markers are known in the art for varying types ofcancer. By way of non-limiting example, cancer cell markers for, e.g.breast cancer cells can include one or more of EPCAM, EGFR, Met,Cadherinll and HER2. The CTCs of a CTC-C are adherent enough that theyassociate even under the conditions of circulating blood. CTC-C can befound associated with white blood cells (WBCs) in circulation. As aconsequence, WBC markers can be occasionally expressed (and/or present)in a CTC-C. As demonstrated herein, CTC-C are much more likely to giverise to a metastasis than, e.g. single CTCs. Accordingly, the presenceof CTC-Cs, or an increased level of CTC-Cs in a subject is indicative ofan increased risk of metastasis.

In one aspect, described herein is an assay comprising measuring thelevel of circulating tumor cell (CTC) clusters in a sample obtained froma subject with a cancer and determining the subject to be at increasedrisk of metastasis of the cancer if the level of CTC clusters isincreased relative to a control level. In one aspect, described hereinis a method of determining if a subject is at increased risk ofmetastasis, the method comprising measuring the level of circulatingtumor cell (CTC) clusters in a sample obtained from a subject with acancer and determining the subject to be at increased risk of metastasisof the cancer if the level of CTC clusters is increased relative to acontrol level. In some embodiments, the cancer is a breast or epithelialcancer. The level of CTC-Cs present in a sample can be measured, e.g. bymeasuring the number of CTC-Cs present and/or by measuring the level ofa marker of CTC-Cs.

The level of CTC-Cs can be measured directly, e.g by detecting thenumber of clusters of tumor cells in a sample. Tumor cells can bedetected, e.g. by immunological methods to detect cells expressing tumorcell surface markers. Non-limiting examples of CTC cell surface markerscan include, EpCAM, EGFR, HER2, CDH11, and/or MET. The tumor cells canbe visualized by microscopy to visually identify clusters, or, forexample, sorted by FACS to detect clusters. In some embodiments, CTC-Ccan be detected using a ^(HB)CTC-Chip, as described in the Examples andin Yu et al. Science 2013 339:580, which is incorporated by referenceherein in its entirety. As a further non-limiting example, a sample,e.g. a blood sample can be subjected to red blood cell lysis and theremaining sample analyzed by a high throughput imagining scanner todetect cell aggregates (e.g. events where the volume/diameter is greaterthan one cell).

In some embodiments, the level of CTC-Cs can be measured by measuringthe expression level of a CTC cluster (CTC-C) marker gene in a sample.In some embodiments, the expression level of more than one marker genecan be determined, e.g. 2 marker genes, 3 marker genes, or more markergenes.

As described herein, the inventors have identified certain genes whichare differentially regulated, to a statistically significant degree, ascompared to a reference level, in CTC-Cs. The identified genes aresometimes referred to herein as marker genes to indicate their relationto being a marker for a CTC-C cell. In some embodiments, a CTC-C markergene can distinguish a CTC-C and/or CTC-C cell from a single CTC.Accordingly, some embodiments of the invention are generally related toassays, methods and systems for assessing the level of CTC-Cs and/or therisk of subject experiencing metastasis. In certain embodiments, theassays and methods are directed to determination and/or measurement ofthe expression level of a gene product (e.g. protein and/or genetranscript such as mRNA) in a biological sample of a subject. In certainembodiments the assays and methods are directed to determination of theexpression level of a gene product of at least two genes in a biologicalsample of a subject, i.e. at least two genes, at least three genes, atleast four genes, at least five genes, at least six genes, at leastseven genes, at least eight genes, at least nine genes, at least 10genes . . . at least 15 genes, . . . at least 25 genes, . . . at least30 genes, or more genes, or any number of genes selected from FIG. 9,Table 2, Table 3, and/or Table 4 as described herein. In someembodiments, the marker gene(s) is selected from the group listed inTable 2, 3, and/or 4. In some embodiments, the assays, methods, andsystems described herein are directed to determination of the expressionlevel of a gene product of at least two genes in a biological sample ofa subject, e.g. at least two genes, or at least three genes, or at leastfour genes, or, e.g. all of the genes of Table 2, 3, and/or 4.

TABLE 2 Exemplary CTC-C marker genes XBP1 ERBB3 KRT19 JUP TACSTD2SERPINB6 CHP1 PSME3 MLPH SSR4 RPS4X RPL32 RGL2 PSMD4 NUCB2 LRPAP1 UBE2L3HSP90AA1 SDHA TUG1 MYL6 AGR2 ELF3 KRT18 ATP5A1 RPL24 EIF3F C20orf24PAPOLA CHCHD2 SNAP23

TABLE 3 Desmosome Marker Genes JUP DSC1 DSC2 DSC3 DSG1 DSG3 DSG4 CTNNB1PKP1 PKP2 PKP3 DSP PLEC EVPL PPL

TABLE 4 Adherence Marker Genes JUP PVRL1 PVRL2 PVRL3 PVRL4 MLLT4 CDH1CDH5 CTNNB1 CTNND1 CTNNA1 CTNNA2 CTNNA3

In some embodiments, a CTC-C marker gene can be plakoglobin. As usedherein, “plakoglobin,” “gamma-catenin,” “junction plakoglobin,” or “JUP”refers to a gene which is known to be common to desmosomes andintermediate junctions. It interacts with cell-cell junction proteinslike, e.g. desmoglein I and E-cadherin. The sequences of plakoglobingenes and gene expression products are known for a number of species,e.g. human plakoglobin (NCBI Gene ID: 3728; mRNA (NCBI Ref Seq:NM_002230; SEQ ID NO: 1); polypeptide (NCBI Ref Seq: NP_002221; SEQ IDNO: 2).

The gene names listed in Table 2, 3 and/or 4 and FIG. 9 are commonnames. NCBI Gene ID numbers for each of the genes listed in Table 2, 3and/or 4 and FIG. 9 can be obtained by searching the “Gene” Database ofthe NCBI (available on the World Wide Web at ncbi.nlm.nih.gov/) usingthe common name as the query and selecting the first returned Homosapiens gene.

In some embodiments, the methods and assays described herein include (a)transforming the gene expression product into a detectable gene target;(b) measuring the amount of the detectable gene target; and (c)comparing the amount of the detectable gene target to an amount of areference, wherein if the amount of the detectable gene target isstatistically significantly different than the amount of the referencelevel, the sample is identified to contain CTC-Cs and/or the subject thesample was obtained from is identified as at risk of developingmetastasis. In some embodiments, if the amount of the detectable genetarget is not statistically significantly different than the amount ofthe reference level, the subject is identified as unlikely to develop ametastasis.

In certain embodiments, the marker gene(s) are selected from the geneslisted in Table 2, 3 and/or 4 and/or FIG. 9. In certain embodiments, oneor more marker genes are selected from the group the genes listed inTable 2, 3 and/or 4.

In subjects who are at risk of metastasis and/or in a cell which is aCTC-C cell, the marker genes listed in Table 2, 3 and/or 4 and/or FIG. 9can be upregulated, e.g. for marker genes listed in Table 2, 3 and/or 4,if the measured marker gene expression in a subject is higher ascompared to a reference level of that marker gene's expression, then thesubject is identified as likely to develop metastasis. Preferably, onelooks at a statistically significant change. However, even if a fewgenes in a group do not differ from normal, e.g. a subject can beidentified as likely to develop metastasis if the overall change of thegroup shows a significant change, preferably a statistically significantchange.

The level of a gene expression product of a marker gene in FIG. 9 and/orTable 2, 3 and/or 4 which is higher than a reference level of thatmarker gene by at least about 10% than the reference amount, at leastabout 20%, at least about 30%, at least about 40%, at least about 50%,at least about 80%, at least about 100%, at least about 200%, at leastabout 300%, at least about 500% or at least about 1000% or more, isindicative that a cell is a CTC-C cell and/or that a subject is at riskof developing metastasis. All possible combinations of 2 or more of theindicated markers are contemplated herein.

As described herein, genes known to be associated with desmosomes and/oradherence junctions are upregulated in CTC-C cells and can serve asbiomarkers thereof. In some embodiments, the level of a gene expressionproduct of a marker gene is a desmosome or adherence junction markergene. The biology of desmosomes and adherence junctions is known in theart, including structural and regulatory genes associated therewith(see, e.g., Kowalczyk and Green. Prog Mol Biol Transl Scie 2013116:95-118; Brooke et al. J Pathol 2012 226:158-171; Delmar et al. CircRes. 2010 107:700-714; Thomason et la. Biochem J 2010 429:419-433;Alberts et al. “Molecular Biology of the Cell” 4^(th) edition, GarlandScience, 2002; and Choi and Weis. HEP 2004 165:23-52; each of which isincorporated by reference herein in its entirety). Non-limiting examplesof desmosome and adherence junction genes are provided in Tables 3 and4, respectively.

As used herein, the term “transforming” or “transformation” refers tochanging an object or a substance, e.g., biological sample, nucleic acidor protein, into another substance. The transformation can be physical,biological or chemical. Exemplary physical transformation includes, butnot limited to, pre-treatment of a biological sample, e.g., from wholeblood to a population of cells or cell groups of a particular size rangeby differential centrifugation or microfluidics sorting. Abiological/chemical transformation can involve at least one enzymeand/or a chemical reagent in a reaction. For example, a DNA sample canbe digested into fragments by one or more restriction enzyme, or anexogenous molecule can be attached to a fragmented DNA sample with aligase. In some embodiments, a DNA sample can undergo enzymaticreplication, e.g., by polymerase chain reaction (PCR).

Methods to measure gene expression products associated with the markergenes described herein are well known to a skilled artisan. Such methodsto measure gene expression products, e.g., protein level, include ELISA(enzyme linked immunosorbent assay), western blot, andimmunoprecipitation, immunofluorescence using detection reagents such asan antibody or protein binding agents. Alternatively, a peptide can bedetected in a subject by introducing into a subject a labeledanti-peptide antibody and other types of detection agent. For example,the antibody can be labeled with a radioactive marker whose presence andlocation in the subject is detected by standard imaging techniques.

For example, antibodies for the polypeptide expression products of themarker genes described herein are commercially available and can be usedfor the purposes of the invention to measure protein expression levels,e.g. anti-plakoglobin (Cat. No. 12083; Abcam; Cambridge, Mass.).Alternatively, since the amino acid sequences for the marker genesdescribed herein are known and publically available at NCBI website, oneof skill in the art can raise their own antibodies against theseproteins of interest for the purpose of the invention. The amino acidsequences of the marker genes described herein have been assigned NCBIaccession numbers for different species such as human, mouse and rat.

In some embodiments, immunohistochemistry (“IHC”) andimmunocytochemistry (“ICC”) techniques can be used. IHC is theapplication of immunochemistry to tissue sections, whereas ICC is theapplication of immunochemistry to cells or tissue imprints after theyhave undergone specific cytological preparations such as, for example,liquid-based preparations. Immunochemistry is a family of techniquesbased on the use of an antibody, wherein the antibodies are used tospecifically target molecules inside or on the surface of cells. Theantibody typically contains a marker that will undergo a biochemicalreaction, and thereby experience a change color, upon encountering thetargeted molecules. In some instances, signal amplification can beintegrated into the particular protocol, wherein a secondary antibody,that includes the marker stain or marker signal, follows the applicationof a primary specific antibody.

In some embodiments, the assay can be a Western blot analysis.Alternatively, proteins can be separated by two-dimensional gelelectrophoresis systems. Two-dimensional gel electrophoresis is wellknown in the art and typically involves iso-electric focusing along afirst dimension followed by SDS-PAGE electrophoresis along a seconddimension. These methods also require a considerable amount of cellularmaterial. The analysis of 2D SDS-PAGE gels can be performed bydetermining the intensity of protein spots on the gel, or can beperformed using immune detection. In other embodiments, protein samplesare analyzed by mass spectroscopy.

Immunological tests can be used with the methods and assays describedherein and include, for example, competitive and non-competitive assaysystems using techniques such as Western blots, radioimmunoassay (RIA),ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, immunodiffusion assays, agglutinationassays, e.g. latex agglutination, complement-fixation assays,immunoradiometric assays, fluorescent immunoassays, e.g. FIA(fluorescence-linked immunoassay), chemiluminescence immunoassays(CLIA), electrochemiluminescence immunoassay (ECLIA, countingimmunoassay (CIA), lateral flow tests or immunoassay (LFIA), magneticimmunoassay (MIA), and protein A immunoassays. Methods for performingsuch assays are known in the art, provided an appropriate antibodyreagent is available. In some embodiment, the immunoassay can be aquantitative or a semi-quantitative immunoassay.

An immunoassay is a biochemical test that measures the concentration ofa substance in a biological sample, typically a fluid sample such asserum, using the interaction of an antibody or antibodies to itsantigen. The assay takes advantage of the highly specific binding of anantibody with its antigen. For the methods and assays described herein,specific binding of the target polypeptides with respective proteins orprotein fragments, or an isolated peptide, or a fusion protein describedherein occurs in the immunoassay to form a target protein/peptidecomplex. The complex is then detected by a variety of methods known inthe art. An immunoassay also often involves the use of a detectionantibody.

Enzyme-linked immunosorbent assay, also called ELISA, enzyme immunoassayor EIA, is a biochemical technique used mainly in immunology to detectthe presence of an antibody or an antigen in a sample. The ELISA hasbeen used as a diagnostic tool in medicine and plant pathology, as wellas a quality control check in various industries.

In one embodiment, an ELISA involving at least one antibody withspecificity for the particular desired antigen (i.e. a marker genepolypeptide as described herein) can also be performed. A known amountof sample and/or antigen is immobilized on a solid support (usually apolystyrene micro titer plate). Immobilization can be eithernon-specific (e.g., by adsorption to the surface) or specific (e.g.where another antibody immobilized on the surface is used to captureantigen or a primary antibody). After the antigen is immobilized, thedetection antibody is added, forming a complex with the antigen. Thedetection antibody can be covalently linked to an enzyme, or can itselfbe detected by a secondary antibody which is linked to an enzyme throughbio-conjugation. Between each step the plate is typically washed with amild detergent solution to remove any proteins or antibodies that arenot specifically bound. After the final wash step the plate is developedby adding an enzymatic substrate to produce a visible signal, whichindicates the quantity of antigen in the sample. Older ELISAs utilizechromogenic substrates, though newer assays employ fluorogenicsubstrates with much higher sensitivity.

In another embodiment, a competitive ELISA is used. Purified antibodiesthat are directed against a target polypeptide or fragment thereof arecoated on the solid phase of multi-well plate, i.e., conjugated to asolid surface. A second batch of purified antibodies that are notconjugated on any solid support is also needed. These non-conjugatedpurified antibodies are labeled for detection purposes, for example,labeled with horseradish peroxidase to produce a detectable signal. Asample (e.g., tumor, blood, serum or urine) from a subject is mixed witha known amount of desired antigen (e.g., a known volume or concentrationof a sample comprising a target polypeptide) together with thehorseradish peroxidase labeled antibodies and the mixture is then areadded to coated wells to form competitive combination. After incubation,if the polypeptide level is high in the sample, a complex of labeledantibody reagent-antigen will form. This complex is free in solution andcan be washed away. Washing the wells will remove the complex. Then thewells are incubated with TMB (3, 3′, 5, 5′-tetramethylbenzidene) colordevelopment substrate for localization of horseradishperoxidase-conjugated antibodies in the wells. There will be no colorchange or little color change if the target polypeptide level is high inthe sample. If there is little or no target polypeptide present in thesample, a different complex in formed, the complex of solid supportbound antibody reagents-target polypeptide. This complex is immobilizedon the plate and is not washed away in the wash step. Subsequentincubation with TMB will produce much color change. Such a competitiveELSA test is specific, sensitive, reproducible and easy to operate.

There are other different forms of ELISA, which are well known to thoseskilled in the art. The standard techniques known in the art for ELISAare described in “Methods in Immunodiagnosis”, 2nd Edition, Rose andBigazzi, eds. John Wiley & Sons, 1980; and Oellerich, M. 1984, J. Clin.Chem. Clin. Biochem. 22:895-904. These references are herebyincorporated by reference in their entirety.

In one embodiment, the levels of a polypeptide in a sample can bedetected by a lateral flow immunoassay test (LFIA), also known as theimmunochromatographic assay, or strip test. LFIAs are a simple deviceintended to detect the presence (or absence) of antigen, e.g. apolypeptide, in a fluid sample. There are currently many LFIA tests areused for medical diagnostics either for home testing, point of caretesting, or laboratory use. LFIA tests are a form of immunoassay inwhich the test sample flows along a solid substrate via capillaryaction. After the sample is applied to the test strip it encounters acolored reagent (generally comprising antibody specific for the testtarget antigen) bound to microparticles which mixes with the sample andtransits the substrate encountering lines or zones which have beenpretreated with another antibody or antigen. Depending upon the level oftarget polypeptides present in the sample the colored reagent can becaptured and become bound at the test line or zone. LFIAs areessentially immunoassays adapted to operate along a single axis to suitthe test strip format or a dipstick format. Strip tests are extremelyversatile and can be easily modified by one skilled in the art fordetecting an enormous range of antigens from fluid samples such asurine, blood, water, and/or homogenized tumor samples etc. Strip testsare also known as dip stick test, the name bearing from the literalaction of “dipping” the test strip into a fluid sample to be tested.LFIA strip tests are easy to use, require minimum training and caneasily be included as components of point-of-care test (POCT)diagnostics to be use on site in the field. LFIA tests can be operatedas either competitive or sandwich assays. Sandwich LFIAs are similar tosandwich ELISA. The sample first encounters colored particles which arelabeled with antibodies raised to the target antigen. The test line willalso contain antibodies to the same target, although it may bind to adifferent epitope on the antigen. The test line will show as a coloredband in positive samples. In some embodiments, the lateral flowimmunoassay can be a double antibody sandwich assay, a competitiveassay, a quantitative assay or variations thereof. Competitive LFIAs aresimilar to competitive ELISA. The sample first encounters coloredparticles which are labeled with the target antigen or an analogue. Thetest line contains antibodies to the target/its analogue. Unlabelledantigen in the sample will block the binding sites on the antibodiespreventing uptake of the colored particles. The test line will show as acolored band in negative samples. There are a number of variations onlateral flow technology. It is also possible to apply multiple capturezones to create a multiplex test.

The use of “dip sticks” or LFIA test strips and other solid supportshave been described in the art in the context of an immunoassay for anumber of antigen biomarkers. U.S. Pat. Nos. 4,943,522; 6,485,982;6,187,598; 5,770,460; 5,622,871; 6,565,808, U.S. patent application Ser.No. 10/278,676; U.S. Ser. No. 09/579,673 and U.S. Ser. No. 10/717,082,which are incorporated herein by reference in their entirety, arenon-limiting examples of such lateral flow test devices. Examples ofpatents that describe the use of “dip stick” technology to detectsoluble antigens via immunochemical assays include, but are not limitedto U.S. Pat. Nos. 4,444,880; 4,305,924; and 4,135,884; which areincorporated by reference herein in their entireties. The apparatusesand methods of these three patents broadly describe a first componentfixed to a solid surface on a “dip stick” which is exposed to a solutioncontaining a soluble antigen that binds to the component fixed upon the“dip stick,” prior to detection of the component-antigen complex uponthe stick. It is within the skill of one in the art to modify theteachings of this “dip stick” technology for the detection ofpolypeptides using antibody reagents as described herein.

Other techniques can be used to detect the level of a polypeptide in asample. One such technique is the dot blot, and adaptation of Westernblotting (Towbin et at., Proc. Nat. Acad. Sci. 76:4350 (1979)). In aWestern blot, the polypeptide or fragment thereof can be dissociatedwith detergents and heat, and separated on an SDS-PAGE gel before beingtransferred to a solid support, such as a nitrocellulose or PVDFmembrane. The membrane is incubated with an antibody reagent specificfor the target polypeptide or a fragment thereof. The membrane is thenwashed to remove unbound proteins and proteins with non-specificbinding. Detectably labeled enzyme-linked secondary or detectionantibodies can then be used to detect and assess the amount ofpolypeptide in the sample tested. The intensity of the signal from thedetectable label corresponds to the amount of enzyme present, andtherefore the amount of polypeptide. Levels can be quantified, forexample by densitometry.

In certain embodiments, the gene expression products as described hereincan be instead determined by determining the level of messenger RNA(mRNA) expression of the marker genes described herein. Such moleculescan be isolated, derived, or amplified from a biological sample, such asa tumor biopsy. Detection of mRNA expression is known by persons skilledin the art, and comprise, for example but not limited to, PCRprocedures, RT-PCR, Northern blot analysis, differential geneexpression, RNA protection assay, microarray analysis, hybridizationmethods, next-generation sequencing etc. Non-limiting examples ofnext-generation sequencing technologies can include Ion Torrent,Illumina, SOLiD, 454; Massively Parallel Signature Sequencingsolid-phase, reversible dye-terminator sequencing; and DNA nanoballsequencing.

In general, the PCR procedure describes a method of gene amplificationwhich is comprised of (i) sequence-specific hybridization of primers tospecific genes or sequences within a nucleic acid sample or library,(ii) subsequent amplification involving multiple rounds of annealing,elongation, and denaturation using a thermostable DNA polymerase, and(iii) screening the PCR products for a band of the correct size. Theprimers used are oligonucleotides of sufficient length and appropriatesequence to provide initiation of polymerization, i.e. each primer isspecifically designed to be complementary to a strand of the genomiclocus to be amplified. In an alternative embodiment, mRNA level of geneexpression products described herein can be determined byreverse-transcription (RT) PCR and by quantitative RT-PCR (QRT-PCR) orreal-time PCR methods. Methods of RT-PCR and QRT-PCR are well known inthe art. The nucleic acid sequences of the marker genes described hereinhave been assigned NCBI accession numbers for different species such ashuman, mouse and rat. Accordingly, a skilled artisan can design anappropriate primer based on the known sequence for determining the mRNAlevel of the respective gene.

Nucleic acid and ribonucleic acid (RNA) molecules can be isolated from aparticular biological sample using any of a number of procedures, whichare well-known in the art, the particular isolation procedure chosenbeing appropriate for the particular biological sample. For example,freeze-thaw and alkaline lysis procedures can be useful for obtainingnucleic acid molecules from solid materials; heat and alkaline lysisprocedures can be useful for obtaining nucleic acid molecules fromurine; and proteinase K extraction can be used to obtain nucleic acidfrom blood (Roiff, A et al. PCR: Clinical Diagnostics and Research,Springer (1994)).

In general, the PCR procedure describes a method of gene amplificationwhich is comprised of (i) sequence-specific hybridization of primers tospecific genes within a nucleic acid sample or library, (ii) subsequentamplification involving multiple rounds of annealing, elongation, anddenaturation using a DNA polymerase, and (iii) screening the PCRproducts for a band of the correct size. The primers used areoligonucleotides of sufficient length and appropriate sequence toprovide initiation of polymerization, i.e. each primer is specificallydesigned to be complementary to each strand of the nucleic acid moleculeto be amplified.

In an alternative embodiment, mRNA level of gene expression productsdescribed herein can be determined by reverse-transcription (RT) PCR andby quantitative RT-PCR (QRT-PCR) or real-time PCR methods. Methods ofRT-PCR and QRT-PCR are well known in the art.

In some embodiments, one or more of the reagents (e.g. an antibodyreagent and/or nucleic acid probe) described herein can comprise adetectable label and/or comprise the ability to generate a detectablesignal (e.g. by catalyzing reaction converting a compound to adetectable product). Detectable labels can comprise, for example, alight-absorbing dye, a fluorescent dye, or a radioactive label.Detectable labels, methods of detecting them, and methods ofincorporating them into reagents (e.g. antibodies and nucleic acidprobes) are well known in the art.

In some embodiments, detectable labels can include labels that can bedetected by spectroscopic, photochemical, biochemical, immunochemical,electromagnetic, radiochemical, or chemical means, such as fluorescence,chemifluoresence, or chemiluminescence, or any other appropriate means.The detectable labels used in the methods described herein can beprimary labels (where the label comprises a moiety that is directlydetectable or that produces a directly detectable moiety) or secondarylabels (where the detectable label binds to another moiety to produce adetectable signal, e.g., as is common in immunological labeling usingsecondary and tertiary antibodies). The detectable label can be linkedby covalent or non-covalent means to the reagent. Alternatively, adetectable label can be linked such as by directly labeling a moleculethat achieves binding to the reagent via a ligand-receptor binding pairarrangement or other such specific recognition molecules. Detectablelabels can include, but are not limited to radioisotopes, bioluminescentcompounds, chromophores, antibodies, chemiluminescent compounds,fluorescent compounds, metal chelates, and enzymes.

In other embodiments, the detection reagent is label with a fluorescentcompound. When the fluorescently labeled antibody is exposed to light ofthe proper wavelength, its presence can then be detected due tofluorescence. In some embodiments, a detectable label can be afluorescent dye molecule, or fluorophore including, but not limited tofluorescein, phycoerythrin, phycocyanin, o-phthaldehyde, fluorescamine,Cy3™, Cy5™, allophycocyanine, Texas Red, peridenin chlorophyll, cyanine,tandem conjugates such as phycoerythrin-Cy5™, green fluorescent protein,rhodamine, fluorescein isothiocyanate (FITC) and Oregon Green™,rhodamine and derivatives (e.g., Texas red and tetrarhodimineisothiocynate (TRITC)), biotin, phycoerythrin, AMCA, CyDyes™,6-carboxyfhiorescein (commonly known by the abbreviations FAM and F),6-carboxy-2′,4′,7′,4,7-hexachlorofiuorescein (HEX),6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfiuorescein (JOE or J),N,N,N′,N′-tetramethyl-6carboxyrhodamine (TAMRA or T),6-carboxy-X-rhodamine (ROX or R), 5-carboxyrhodamine-6G (R6G5 or G5),6-carboxyrhodamine-6G (R6G6 or G6), and rhodamine 110; cyanine dyes,e.g. Cy3, Cy5 and Cy7 dyes; coumarins, e.g umbelliferone; benzimidedyes, e.g. Hoechst 33258; phenanthridine dyes, e.g. Texas Red; ethidiumdyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes;polymethine dyes, e.g. cyanine dyes such as Cy3, Cy5, etc; BODIPY dyesand quinoline dyes. In some embodiments, a detectable label can be aradiolabel including, but not limited to 3H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P, and³³P. In some embodiments, a detectable label can be an enzyme including,but not limited to horseradish peroxidase and alkaline phosphatase. Anenzymatic label can produce, for example, a chemiluminescent signal, acolor signal, or a fluorescent signal. Enzymes contemplated for use todetectably label an antibody reagent include, but are not limited to,malate dehydrogenase, staphylococcal nuclease, delta-V-steroidisomerase, yeast alcohol dehydrogenase, alpha-glycerophosphatedehydrogenase, triose phosphate isomerase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase,glucoamylase and acetylcholinesterase. In some embodiments, a detectablelabel is a chemiluminescent label, including, but not limited tolucigenin, luminol, luciferin, isoluminol, theromatic acridinium ester,imidazole, acridinium salt and oxalate ester. In some embodiments, adetectable label can be a spectral colorimetric label including, but notlimited to colloidal gold or colored glass or plastic (e.g.,polystyrene, polypropylene, and latex) beads.

In some embodiments, detection reagents can also be labeled with adetectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin.Other detection systems can also be used, for example, abiotin-streptavidin system. In this system, the antibodiesimmunoreactive (i. e. specific for) with the biomarker of interest isbiotinylated. Quantity of biotinylated antibody bound to the biomarkeris determined using a streptavidin-peroxidase conjugate and achromagenic substrate. Such streptavidin peroxidase detection kits arecommercially available, e. g. from DAKO; Carpinteria, Calif. A reagentcan also be detectably labeled using fluorescence emitting metals suchas ¹⁵²Eu, or others of the lanthanide series. These metals can beattached to the reagent using such metal chelating groups asdiethylenetriaminepentaacetic acid (DTPA) or ethylene diaminetetraaceticacid (EDTA).

In some embodiments, the expression level of a CTC-C marker gene in ablood sample is measured. In some embodiments, the expression level of aCTC-C marker gene in circulating tumor cells in the sample is measured.In some embodiments, the expression level of a CTC-C marker gene inCTC-Cs in the sample is measured. CTCs and CTC-Cs can be isolated asdescribed above herein, e.g. using an HB-CTC-Chip or FACS. In someembodiments, the expression level of a CTC-C marker gene in cancer cellsobtained from the subject is measured, e.g. the expression level in atumor sample can be measured.

In some embodiments of any of the aspects described herein, the level ofexpression products of more than one gene can be determinedsimultaneously (e.g. a multiplex assay) or in parallel. In someembodiments, the level of expression products of no more than 200 othergenes is determined. In some embodiments, the level of expressionproducts of no more than 100 other genes is determined. In someembodiments, the level of expression products of no more than 20 othergenes is determined. In some embodiments, the level of expressionproducts of no more than 10 other genes is determined.

In some embodiments, the reference can be a level of expression of themarker gene product in a population of subjects who have beendemonstrated to not be at risk for metastasis. In some embodiments, thereference can be a level of expression of the marker gene product in aCTC or a population of CTCs not isolated from CTC-Cs. In someembodiments, the reference can also be a level of expression of themarker gene product in a control sample, a pooled sample of controlindividuals or a numeric value or range of values based on the same.

In some embodiments, an increased level of CTC-Cs is a level at least1.5× greater than the control level, e.g. 1.5× or greater, 2× orgreater, 2.5× or greater, 3× or greater, 4× or greater, 5× or greater,10× or greater, or more. In some embodiments, an increased level of aCTC-C marker gene is a level at least 1.5× greater than the controllevel, e.g. 1.5× or greater, 2× or greater, 2.5× or greater, 3× orgreater, 4× or greater, 5× or greater, 10× or greater, or more.

The term “sample” or “test sample” as used herein denotes a sample takenor isolated from a biological organism, e.g., a blood sample from asubject. Exemplary biological samples include, but are not limited to, abiofluid sample; serum; plasma; urine; saliva; a tumor sample; a tumorbiopsy and/or tissue sample etc. The term also includes a mixture of theabove-mentioned samples. The term “test sample” also includes untreatedor pretreated (or pre-processed) biological samples. In someembodiments, a test sample can comprise cells from subject. In someembodiments, a test sample can be a tumor cell test sample, e.g. thesample can comprise cancerous cells, cells from a tumor, and/or a tumorbiopsy. In some embodiments, the test sample can be a blood sample. Insome embodiments, the test sample can be a serum sample.

The test sample can be obtained by removing a sample of cells from asubject, but can also be accomplished by using previously isolated cells(e.g. isolated at a prior timepoint and isolated by the same or anotherperson). In addition, the test sample can be freshly collected or apreviously collected sample.

In some embodiments, the test sample can be an untreated test sample. Asused herein, the phrase “untreated test sample” refers to a test samplethat has not had any prior sample pre-treatment except for dilutionand/or suspension in a solution. Exemplary methods for treating a testsample include, but are not limited to, centrifugation, filtration,sonication, homogenization, heating, freezing and thawing, andcombinations thereof. In some embodiments, the test sample can be afrozen test sample, e.g., a frozen tissue. The frozen sample can bethawed before employing methods, assays and systems described herein.After thawing, a frozen sample can be centrifuged before being subjectedto methods, assays and systems described herein. In some embodiments,the test sample is a clarified test sample, for example, bycentrifugation and collection of a supernatant comprising the clarifiedtest sample. In some embodiments, a test sample can be a pre-processedtest sample, for example, supernatant or filtrate resulting from atreatment selected from the group consisting of centrifugation,filtration, thawing, purification, and any combinations thereof. In someembodiments, the test sample can be treated with a chemical and/orbiological reagent. Chemical and/or biological reagents can be employedto protect and/or maintain the stability of the sample, includingbiomolecules (e.g., nucleic acid and protein) therein, duringprocessing. One exemplary reagent is a protease inhibitor, which isgenerally used to protect or maintain the stability of protein duringprocessing. The skilled artisan is well aware of methods and processesappropriate for pre-processing of biological samples required fordetermination of the level of an expression product as described herein.

As demonstrated herein, the increased expression of CTC-C marker genescontributes to the metastatic potential of these cells. Accordingly, thelevel of CTC-Cs, and/or the metastatic potential of the CTC-Cs can bereduced by inhibiting the expression and/or activity of one or moreCTC-C marker genes. In one aspect, described herein is a method ofreducing the level of circulating tumor cell (CTC) clusters in a subjectwith cancer, the method comprising reducing the level of expression oractivity of a CTC-C marker gene. In one aspect, described herein is amethod of treating cancer metastasis, the method comprising reducing thelevel of expression or activity of a CTC-C marker gene.

As used herein, the term “inhibitor” refers to an agent which candecrease the expression and/or activity of the targeted expressionproduct (e.g. mRNA encoding the target or a target polypeptide), e.g. byat least 10% or more, e.g. by 10% or more, 50% or more, 70% or more, 80%or more, 90% or more, 95% or more, or 98% or more. The efficacy of aninhibitor of, for example, plakoglobin, e.g. its ability to decrease thelevel and/or activity of plakoglobin can be determined, e.g. bymeasuring the level of an expression product of plakoglobin and/or theactivity of plakoglobin. Methods for measuring the level of a given mRNAand/or polypeptide are known to one of skill in the art, e.g. RTPCR withprimers can be used to determine the level of RNA and Western blottingwith an antibody (e.g. an anti-JUP antibody, e.g. Cat No. ab12083;Abcam; Cambridge, Mass.) can be used to determine the level of apolypeptide. The activity of, e.g. plakoglobin can be determined usingmethods known in the art and described below herein, e.g. the ability ofCTC-Cs to form new tumors, e.g. metastaize. In some embodiments, theinhibitor of JUP can be an inhibitory nucleic acid; an aptamer; anantibody reagent; an antibody; or a small molecule.

In some embodiments, reducing the level of expression or activity of aCTC-C marker gene comprises administering a CTC-C marker gene inhibitorynucleic acid. In some embodiments, the inhibitory nucleic acid is asiRNA. In some embodiments, the CTC-C marker gene is plakoglobin.

In one aspect, described herein is a method of treating cancer, themethod comprising measuring the level of circulating tumor cell (CTC)clusters in a sample obtained from a subject; administering a treatmentto prevent or reduce metastasis if the level of CTC clusters isincreased relative to a control level; and not administering a treatmentto prevent or reduce metastasis if the level of CTC clusters is notincreased relative to a control level. In some embodiments, the cancercan be breast and/or epithelial cancer.

Treatments to prevent and/or reduce metastasis are known to one of skillin the art. Non-limiting examples of such treatments can includechemotherapy, radiation therapy, removal of a tumor (e.g. surgicalremoval), and/or administration of an inhibitor of a CTC-C marker geneas described elsewhere herein. In some embodiments, not administering atreatment can comprise a clinical approach of monitoring withouttherapeutic intervention, e.g. “watchful waiting.”

In some embodiments, the methods, assays, and systems described hereincan further comprise a step of obtaining a test sample from a subject.In some embodiments, the subject is a human subject. In someembodiments, the subject is a subject having or diagnosed as havingcancer. In some embodiments, the subject is a subject in need oftreatment for cancer.

In some embodiments, the methods described herein relate to treating asubject having or diagnosed as having cancer. In some embodiments thecancer can be breast cancer and/or epithelial cancer. Subjects havingcancer can be identified by a physician using current methods ofdiagnosing cancer. Symptoms and/or complications of cancer whichcharacterize these conditions and aid in diagnosis are well known in theart and include but are not limited to, e.g. for breast cancer, lumps,inflammation, itching, changes in skin appearance and/or texture, pain,discharge, and/or swelling. Tests that may aid in a diagnosis of, e.g.breast cancer include, but are not limited to, mammograms and biopsies.A family history of breast cancer or exposure to risk factors for breastcancer can also aid in determining if a subject is likely to have canceror in making a diagnosis of cancer.

The compositions and methods described herein can be administered to asubject having or diagnosed as having cancer. In some embodiments, themethods described herein comprise administering an effective amount ofcompositions described herein, e.g. an inhibitor of a CTC-C marker geneto a subject in order to alleviate a symptom of a cancer. As usedherein, “alleviating a symptom of a cancer” is ameliorating anycondition or symptom associated with the cancer. As compared with anequivalent untreated control, such reduction is by at least 5%, 10%,20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by anystandard technique. A variety of means for administering thecompositions described herein to subjects are known to those of skill inthe art. Such methods can include, but are not limited to oral,parenteral, intravenous, intramuscular, subcutaneous, transdermal,airway (aerosol), pulmonary, cutaneous, topical, injection, orintratumoral administration. Administration can be local or systemic.

The term “effective amount” as used herein refers to the amount of aninhibitor of a CTC-C marker gene needed to alleviate at least one ormore symptom of the disease or disorder, and relates to a sufficientamount of pharmacological composition to provide the desired effect. Theterm “therapeutically effective amount” therefore refers to an amount ofan inhibitor of a CTC-C marker gene that is sufficient to provide aparticular anti-cancer effect when administered to a typical subject. Aneffective amount as used herein, in various contexts, would also includean amount sufficient to delay the development of a symptom of thedisease, alter the course of a symptom disease (for example but notlimited to, slowing the progression of a symptom of the disease), orreverse a symptom of the disease. Thus, it is not generally practicableto specify an exact “effective amount”. However, for any given case, anappropriate “effective amount” can be determined by one of ordinaryskill in the art using only routine experimentation.

Effective amounts, toxicity, and therapeutic efficacy can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dosage can vary depending upon the dosage formemployed and the route of administration utilized. The dose ratiobetween toxic and therapeutic effects is the therapeutic index and canbe expressed as the ratio LD50/ED50. Compositions and methods thatexhibit large therapeutic indices are preferred. A therapeuticallyeffective dose can be estimated initially from cell culture assays.Also, a dose can be formulated in animal models to achieve a circulatingplasma concentration range that includes the IC50 (i.e., theconcentration of an inhibitor of a CTC-C marker gene, which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture, orin an appropriate animal model. Levels in plasma can be measured, forexample, by high performance liquid chromatography. The effects of anyparticular dosage can be monitored by a suitable bioassay, e.g., assayfor cancer growth, survival, and/or metastasis among others. The dosagecan be determined by a physician and adjusted, as necessary, to suitobserved effects of the treatment.

In some embodiments, the technology described herein relates to apharmaceutical composition comprising an inhibitor of a CTC-C markergene as described herein, and optionally a pharmaceutically acceptablecarrier. Pharmaceutically acceptable carriers and diluents includesaline, aqueous buffer solutions, solvents and/or dispersion media. Theuse of such carriers and diluents is well known in the art. Somenon-limiting examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, methylcellulose, ethyl cellulose,microcrystalline cellulose and cellulose acetate; (4) powderedtragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such asmagnesium stearate, sodium lauryl sulfate and talc; (8) excipients, suchas cocoa butter and suppository waxes; (9) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; (22) C₂-C₁₂ alcohols, such as ethanol; and (23)other non-toxic compatible substances employed in pharmaceuticalformulations. Wetting agents, coloring agents, release agents, coatingagents, sweetening agents, flavoring agents, perfuming agents,preservative and antioxidants can also be present in the formulation.The terms such as “excipient”, “carrier”, “pharmaceutically acceptablecarrier” or the like are used interchangeably herein. In someembodiments, the carrier inhibits the degradation of the active agent,e.g. an inhibitor of a CTC-C marker gene as described herein.

In some embodiments, the pharmaceutical composition comprising aninhibitor of a CTC-C marker gene as described herein can be a parenteraldose form. Since administration of parenteral dosage forms typicallybypasses the patient's natural defenses against contaminants, parenteraldosage forms are preferably sterile or capable of being sterilized priorto administration to a patient. Examples of parenteral dosage formsinclude, but are not limited to, solutions ready for injection, dryproducts ready to be dissolved or suspended in a pharmaceuticallyacceptable vehicle for injection, suspensions ready for injection, andemulsions. In addition, controlled-release parenteral dosage forms canbe prepared for administration of a patient, including, but not limitedto, DUROS®-type dosage forms and dose-dumping.

Suitable vehicles that can be used to provide parenteral dosage forms ofan inhibitor of a CTC-C marker gene as disclosed within are well knownto those skilled in the art. Examples include, without limitation:sterile water; water for injection USP; saline solution; glucosesolution; aqueous vehicles such as but not limited to, sodium chlorideinjection, Ringer's injection, dextrose Injection, dextrose and sodiumchloride injection, and lactated Ringer's injection; water-misciblevehicles such as, but not limited to, ethyl alcohol, polyethyleneglycol, and propylene glycol; and non-aqueous vehicles such as, but notlimited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyloleate, isopropyl myristate, and benzyl benzoate. Compounds that alteror modify the solubility of a pharmaceutically acceptable salt of aninhibitor of a CTC-C marker gene as disclosed herein can also beincorporated into the parenteral dosage forms of the disclosure,including conventional and controlled-release parenteral dosage forms.

Pharmaceutical compositions comprising an inhibitor of a CTC-C markergene can also be formulated to be suitable for oral administration, forexample as discrete dosage forms, such as, but not limited to, tablets(including without limitation scored or coated tablets), pills, caplets,capsules, chewable tablets, powder packets, cachets, troches, wafers,aerosol sprays, or liquids, such as but not limited to, syrups, elixirs,solutions or suspensions in an aqueous liquid, a non-aqueous liquid, anoil-in-water emulsion, or a water-in-oil emulsion. Such compositionscontain a predetermined amount of the pharmaceutically acceptable saltof the disclosed compounds, and may be prepared by methods of pharmacywell known to those skilled in the art. See generally, Remington: TheScience and Practice of Pharmacy, 21st Ed., Lippincott, Williams, andWilkins, Philadelphia Pa. (2005).

Conventional dosage forms generally provide rapid or immediate drugrelease from the formulation. Depending on the pharmacology andpharmacokinetics of the drug, use of conventional dosage forms can leadto wide fluctuations in the concentrations of the drug in a patient'sblood and other tissues. These fluctuations can impact a number ofparameters, such as dose frequency, onset of action, duration ofefficacy, maintenance of therapeutic blood levels, toxicity, sideeffects, and the like. Advantageously, controlled-release formulationscan be used to control a drug's onset of action, duration of action,plasma levels within the therapeutic window, and peak blood levels. Inparticular, controlled- or extended-release dosage forms or formulationscan be used to ensure that the maximum effectiveness of a drug isachieved while minimizing potential adverse effects and safety concerns,which can occur both from under-dosing a drug (i.e., going below theminimum therapeutic levels) as well as exceeding the toxicity level forthe drug. In some embodiments, the an inhibitor of a CTC-C marker genecan be administered in a sustained release formulation.

Controlled-release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non-controlledrelease counterparts. Ideally, the use of an optimally designedcontrolled-release preparation in medical treatment is characterized bya minimum of drug substance being employed to cure or control thecondition in a minimum amount of time. Advantages of controlled-releaseformulations include: 1) extended activity of the drug; 2) reduceddosage frequency; 3) increased patient compliance; 4) usage of lesstotal drug; 5) reduction in local or systemic side effects; 6)minimization of drug accumulation; 7) reduction in blood levelfluctuations; 8) improvement in efficacy of treatment; 9) reduction ofpotentiation or loss of drug activity; and 10) improvement in speed ofcontrol of diseases or conditions. Kim, Cherng-ju, Controlled ReleaseDosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).

Most controlled-release formulations are designed to initially releasean amount of drug (active ingredient) that promptly produces the desiredtherapeutic effect, and gradually and continually release other amountsof drug to maintain this level of therapeutic or prophylactic effectover an extended period of time. In order to maintain this constantlevel of drug in the body, the drug must be released from the dosageform at a rate that will replace the amount of drug being metabolizedand excreted from the body. Controlled-release of an active ingredientcan be stimulated by various conditions including, but not limited to,pH, ionic strength, osmotic pressure, temperature, enzymes, water, andother physiological conditions or compounds.

A variety of known controlled- or extended-release dosage forms,formulations, and devices can be adapted for use with the salts andcompositions of the disclosure. Examples include, but are not limitedto, those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809;3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548;5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 B1; each ofwhich is incorporated herein by reference. These dosage forms can beused to provide slow or controlled-release of one or more activeingredients using, for example, hydroxypropylmethyl cellulose, otherpolymer matrices, gels, permeable membranes, osmotic systems (such asOROS® (Alza Corporation, Mountain View, Calif. USA)), or a combinationthereof to provide the desired release profile in varying proportions.

The methods described herein can further comprise administering a secondagent and/or treatment to the subject, e.g. as part of a combinatorialtherapy. A second agent and/or treatment can include a chemotherapyand/or radiation therapy, and/or surgery.

As used herein, a “chemotherapy” refers to a substance that reduces ordecreases the growth, survival, and/or metastasis of cancer cells.Chemotherapies can include toxins, small molecules, and/or polypeptides.Non-limiting examples of a second agent and/or treatment can includeradiation therapy, surgery, gemcitabine, cisplastin, paclitaxel,carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide,rapamycin, ABT-737, PI-103; alkylating agents such as thiotepa andCYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan,improsulfan and piposulfan; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gammalI and calicheamicinomegaIl (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994));dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antiobiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE®Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin, oxaliplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; NAVELBINE™ vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar,CPT-11) (including the treatment regimen of irinotecan with 5-FU andleucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; capecitabine; combretastatin;leucovorin (LV); oxaliplatin, including the oxaliplatin treatmentregimen (FOLFOX); lapatinib (Tykerb™); inhibitors of PKC-alpha, Raf,H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF-A that reduce cellproliferation and pharmaceutically acceptable salts, acids orderivatives of any of the above.

In addition, the methods of treatment can further include the use ofradiation or radiation therapy. Further, the methods of treatment canfurther include the use of surgical treatments.

In certain embodiments, an effective dose of a composition comprising aninhibitor of a CTC-C marker gene as described herein can be administeredto a patient once. In certain embodiments, an effective dose of acomposition comprising an inhibitor of a CTC-C marker gene can beadministered to a patient repeatedly. For systemic administration,subjects can be administered a therapeutic amount of a compositioncomprising an inhibitor of a CTC-C marker gene, such as, e.g. 0.1 mg/kg,0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg,20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, or more.

In some embodiments, after an initial treatment regimen, the treatmentscan be administered on a less frequent basis. For example, aftertreatment biweekly for three months, treatment can be repeated once permonth, for six months or a year or longer. Treatment according to themethods described herein can reduce levels of a marker or symptom of acondition, e.g. CTC-C levels by at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80% or at least 90% or more.

The dosage of a composition as described herein can be determined by aphysician and adjusted, as necessary, to suit observed effects of thetreatment. With respect to duration and frequency of treatment, it istypical for skilled clinicians to monitor subjects in order to determinewhen the treatment is providing therapeutic benefit, and to determinewhether to increase or decrease dosage, increase or decreaseadministration frequency, discontinue treatment, resume treatment, ormake other alterations to the treatment regimen. The dosing schedule canvary from once a week to daily depending on a number of clinicalfactors, such as the subject's sensitivity to an inhibitor of a CTC-Cmarker gene. The desired dose or amount of activation can beadministered at one time or divided into subdoses, e.g., 2-4 subdosesand administered over a period of time, e.g., at appropriate intervalsthrough the day or other appropriate schedule. In some embodiments,administration can be chronic, e.g., one or more doses and/or treatmentsdaily over a period of weeks or months. Examples of dosing and/ortreatment schedules are administration daily, twice daily, three timesdaily or four or more times daily over a period of 1 week, 2 weeks, 3weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6months, or more. A composition comprising an inhibitor of a CTC-C markergene can be administered over a period of time, such as over a 5 minute,10 minute, 15 minute, 20 minute, or 25 minute period.

The dosage ranges for the administration of an inhibitor of a CTC-Cmarker gene, according to the methods described herein depend upon, forexample, the form of the inhibitor of a CTC-C marker gene, its potency,and the extent to which symptoms, markers, or indicators of a conditiondescribed herein are desired to be reduced, for example the percentagereduction desired for symptoms (e.g. CTC-C levels). The dosage shouldnot be so large as to cause adverse side effects. Generally, the dosagewill vary with the age, condition, and sex of the patient and can bedetermined by one of skill in the art. The dosage can also be adjustedby the individual physician in the event of any complication.

The efficacy of an inhibitor of a CTC-C marker gene in, e.g. thetreatment of a condition described herein, or to induce a response asdescribed herein (e.g. a reduction of CTC-C levels) can be determined bythe skilled clinician. However, a treatment is considered “effectivetreatment,” as the term is used herein, if one or more of the signs orsymptoms of a condition described herein are altered in a beneficialmanner, other clinically accepted symptoms are improved, or evenameliorated, or a desired response is induced e.g., by at least 10%following treatment according to the methods described herein. Efficacycan be assessed, for example, by measuring a marker, indicator, symptom,and/or the incidence of a condition treated according to the methodsdescribed herein or any other measurable parameter appropriate, e.g.metastasis and/or CTC-C levels. Efficacy can also be measured by afailure of an individual to worsen as assessed by hospitalization, orneed for medical interventions (i.e., progression of the disease ishalted). Methods of measuring these indicators are known to those ofskill in the art and/or are described herein. Treatment includes anytreatment of a disease in an individual or an animal (some non-limitingexamples include a human or an animal) and includes: (1) inhibiting thedisease, e.g., preventing a worsening of symptoms (e.g. pain orinflammation); or (2) relieving the severity of the disease, e.g.,causing regression of symptoms. An effective amount for the treatment ofa disease means that amount which, when administered to a subject inneed thereof, is sufficient to result in effective treatment as thatterm is defined herein, for that disease. Efficacy of an agent can bedetermined by assessing physical indicators of a condition or desiredresponse, (e.g. metastasis). It is well within the ability of oneskilled in the art to monitor efficacy of administration and/ortreatment by measuring any one of such parameters, or any combination ofparameters. Efficacy can be assessed in animal models of a conditiondescribed herein, for example treatment of cancer in mouse models. Whenusing an experimental animal model, efficacy of treatment is evidencedwhen a statistically significant change in a marker is observed, e.g.metastasis and/or CTC-C levels.

In vitro and animal model assays are provided herein which allow theassessment of a given dose of an inhibitor of a CTC-C marker gene. Byway of non-limiting example, the effects of a dose of an inhibitor of aCTC-C marker gene can be assessed by measuring the level of CTC-Cs ascompared to CTCs and/or normal cancer cells. A non-limiting example of aprotocol for such an assay is as follows: Cancer cells are subjected toa Vybrant™ cell-to-cell adhesion assay in the presence or absence of theinhibitor of a CTC-C marker gene and the adhesion determined.

The efficacy of a given dosage combination can also be assessed in ananimal model, e.g. a mouse model of cancer. For example, cancer cellscan be injected into the mammary fat pad of immunodeficient mice, aninhibitor of a CTC-C marker gene administered and tumor growth (and/orCTC-C levels) measured.

A kit is any manufacture (e.g., a package or container) comprising atleast one reagent, e.g., an antibody reagent(s), for specificallydetecting and/or measuring the level of CTC-Cs in a sample, themanufacture being promoted, distributed, or sold as a unit forperforming the methods or assays described herein. When the kits, andmethods described herein are used for diagnosis and/or treatment of acancer (e.g. breast cancer), the CTC-C detection probes or systems canbe selected such that a positive result is obtained in at least about20%, at least about 40%, at least about 60%, at least about 80%, atleast about 90%, at least about 95%, at least about 99% or in 100% ofsubjects having elevated levels of CTC-Cs, e.g. increased risk ofmetastasis.

In some embodiments, described herein is a kit for the detection ofCTC-Cs in a sample, the kit comprising at least a first antibody reagentas described herein which specifically binds a CTC-C marker gene, e.g.JUP, on a solid support and comprising a detectable label. In someembodiments, the kit can further comprise at least a second antibodyreagent as described herein which specifically binds a CTC-C markergene, wherein the first and second antibody reagents can bindsimultaneously to a single polypeptide molecule. In some embodiments,the antibody reagent(s) can be configured to permit sandwich immunoassaydetection of a marker gene polypeptide present in a sample. In someembodiments, a sandwich immunoassay can comprise an ELISA, lateral flowimmunoassay, fluorescence immunoassay, a dipstick immunoassay, a urinedipstick immunoassay, or the like.

In some embodiments, described herein is a kit for the detection ofCTC-Cs in a sample, the kit comprising at least a first nucleic acidprimer and/or probe which specifically binds a CTC-C marker gene, e.g.JUP. In some embodiments, the primer and/or probe can further comprise adetectable level and/or be conjugated to a solid support

When the expression level of a CTC-C marker gene is used in the methodsand assays described herein, the expression level of the gene can becompared with the expression level of the marker in non-canceroussamples of the same type or to another reference value or referencestandard as described herein.

The kits described herein can optionally comprise additional componentsuseful for performing the methods and assays described herein. By way ofexample, the kit can comprise fluids (e.g., buffers) suitable forbinding an probe with a target with which it specifically binds, one ormore sample compartments, an instructional material which describesperformance of a method as described herein, a sample of blood (e.g. asa reference), and the like. A kit can further comprise devices and/orreagents for concentrating a target in a sample, e.g. a blood sample.

Preferably, a diagnostic kit for use with the methods and assaysdisclosed herein contains detection reagents for CTC-Cs and/or CTC-Cmarker gene expression products. Such detection reagents comprise inaddition to reagents specific for the target, for example, buffersolutions, labels or washing liquids etc. Furthermore, the kit cancomprise an amount of a known target, which can be used for acalibration of the kit or as an internal control. A diagnostic kit forthe can also comprise accessory ingredients like secondary affinityligands, e.g., secondary antibodies, detection dyes and any othersuitable compound or liquid necessary for the performance of a detectionmethod known to the person skilled in the art. Such ingredients areknown to the person skilled in the art and may vary depending on thedetection method carried out. Additionally, the kit may comprise aninstruction leaflet and/or may provide information as to the relevanceof the obtained results.

For convenience, the meaning of some terms and phrases used in thespecification, examples, and appended claims, are provided below. Unlessstated otherwise, or implicit from context, the following terms andphrases include the meanings provided below. The definitions areprovided to aid in describing particular embodiments, and are notintended to limit the claimed invention, because the scope of theinvention is limited only by the claims. Unless otherwise defined, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. If there is an apparent discrepancy between the usageof a term in the art and its definition provided herein, the definitionprovided within the specification shall prevail.

For convenience, certain terms employed herein, in the specification,examples and appended claims are collected here.

The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all usedherein to mean a decrease by a statistically significant amount. In someembodiments, “reduce,” “reduction” or “decrease” or “inhibit” typicallymeans a decrease by at least 10% as compared to a reference level (e.g.the absence of a given treatment) and can include, for example, adecrease by at least about 10%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or more. As used herein,“reduction” or “inhibition” does not encompass a complete inhibition orreduction as compared to a reference level. “Complete inhibition” is a100% inhibition as compared to a reference level. A decrease can bepreferably down to a level accepted as within the range of normal for anindividual without a given disorder.

The terms “increased”, “increase”, “enhance”, or “activate” are all usedherein to mean an increase by a statically significant amount. In someembodiments, the terms “increased”, “increase”, “enhance”, or “activate”can mean an increase of at least 10% as compared to a reference level,for example an increase of at least about 20%, or at least about 30%, orat least about 40%, or at least about 50%, or at least about 60%, or atleast about 70%, or at least about 80%, or at least about 90% or up toand including a 100% increase or any increase between 10-100% ascompared to a reference level, or at least about a 2-fold, or at leastabout a 3-fold, or at least about a 4-fold, or at least about a 5-foldor at least about a 10-fold increase, or any increase between 2-fold and10-fold or greater as compared to a reference level. In the context of amarker or symptom, a “increase” is a statistically significant increasein such level.

As used herein, a “subject” means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g.,chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. Insome embodiments, the subject is a mammal, e.g., a primate, e.g., ahuman. The terms, “individual,” “patient” and “subject” are usedinterchangeably herein.

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but is notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models of cancer.A subject can be male or female.

A subject can be one who has been previously diagnosed with oridentified as suffering from or having a condition in need of treatment(e.g. cancer) or one or more complications related to such a condition,and optionally, have already undergone treatment for cancer or the oneor more complications related to cancer. Alternatively, a subject canalso be one who has not been previously diagnosed as having cancer orone or more complications related to cancer. For example, a subject canbe one who exhibits one or more risk factors for cancer or one or morecomplications related to cancer or a subject who does not exhibit riskfactors.

A “subject in need” of treatment for a particular condition can be asubject having that condition, diagnosed as having that condition, or atrisk of developing that condition.

As used herein, the term “cancer” or “tumor” refers to an uncontrolledgrowth of cells which interferes with the normal functioning of thebodily organs and systems. A subject who has a cancer or a tumor is asubject having objectively measurable cancer cells present in thesubject's body. Included in this definition are benign and malignantcancers, as well as dormant tumors or micrometastases. Cancers whichmigrate from their original location and seed vital organs caneventually lead to the death of the subject through the functionaldeterioration of the affected organs. Epithelial cancers can be, e.g.,selected from the group consisting of: carcinoma; adenocarcinoma; basalcell carcinoma; squamous cell carcinoma; large cell carcinoma; smallcell carcinoma; colorectal adenocarcinoma; lung cancer; breast cancer;prostate cancer; colon cancer; rectal cancer; pancreatic cancer; kidneycancer; ovarian cancer; stomach cancer; intestinal cancer; oral cancer;esophageal cancer; lip cancer; bladder cancer; cervical cancer; skincancer; hepatocellular carcinoma; and renal cell carcinoma.

As used herein, “expression level” refers to the number of mRNAmolecules and/or polypeptide molecules encoded by a given gene that arepresent in a cell or sample. Expression levels can be increased ordecreased relative to a reference level.

The term “agent” refers generally to any entity which is normally notpresent or not present at the levels being administered to a cell,tissue or subject. An agent can be selected from a group including butnot limited to: polynucleotides; polypeptides; small molecules; andantibodies or antigen-binding fragments thereof. A polynucleotide can beRNA or DNA, and can be single or double stranded, and can be selectedfrom a group including, for example, nucleic acids and nucleic acidanalogues that encode a polypeptide. A polypeptide can be, but is notlimited to, a naturally-occurring polypeptide, a mutated polypeptide ora fragment thereof that retains the function of interest. Furtherexamples of agents include, but are not limited to a nucleic acidaptamer, peptide-nucleic acid (PNA), locked nucleic acid (LNA), smallorganic or inorganic molecules; saccharide; oligosaccharides;polysaccharides; biological macromolecules, peptidomimetics; nucleicacid analogs and derivatives; extracts made from biological materialssuch as bacteria, plants, fungi, or mammalian cells or tissues andnaturally occurring or synthetic compositions. An agent can be appliedto the media, where it contacts the cell and induces its effects.Alternatively, an agent can be intracellular as a result of introductionof a nucleic acid sequence encoding the agent into the cell and itstranscription resulting in the production of the nucleic acid and/orprotein environmental stimuli within the cell. In some embodiments, theagent is any chemical, entity or moiety, including without limitationsynthetic and naturally-occurring non-proteinaceous entities. In certainembodiments the agent is a small molecule having a chemical moietyselected, for example, from unsubstituted or substituted alkyl,aromatic, or heterocyclyl moieties including macrolides, leptomycins andrelated natural products or analogues thereof. Agents can be known tohave a desired activity and/or property, or can be selected from alibrary of diverse compounds. As used herein, the term “small molecule”can refer to compounds that are “natural product-like,” however, theterm “small molecule” is not limited to “natural product-like”compounds. Rather, a small molecule is typically characterized in thatit contains several carbon-carbon bonds, and has a molecular weight morethan about 50, but less than about 5000 Daltons (5 kD). Preferably thesmall molecule has a molecular weight of less than 3 kD, still morepreferably less than 2 kD, and most preferably less than 1 kD. In somecases it is preferred that a small molecule have a molecular mass equalto or less than 700 Daltons.

As used herein the term “chemotherapeutic agent” refers to any chemicalor biological agent with therapeutic usefulness in the treatment ofdiseases characterized by abnormal cell growth. Such diseases includetumors, neoplasms and cancer as well as diseases characterized byhyperplastic growth. These agents can function to inhibit a cellularactivity upon which the cancer cell depends for continued proliferation.In some aspect of all the embodiments, a chemotherapeutic agent is acell cycle inhibitor or a cell division inhibitor. Categories ofchemotherapeutic agents that are useful in the methods of the inventioninclude alkylating/alkaloid agents, antimetabolites, hormones or hormoneanalogs, and miscellaneous antineoplastic drugs. Most of these agentsare directly or indirectly toxic to cancer cells. In one embodiment, achemotherapeutic agent is a radioactive molecule. One of skill in theart can readily identify a chemotherapeutic agent of use (e.g. seeSlapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison'sPrinciples of Internal Medicine, 14th edition; Perry et al.,Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2nd ed. 2000Churchill Livingstone, Inc; Baltzer L, Berkery R (eds): Oncology PocketGuide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; FischerD S, Knobf M F, Durivage H J (eds): The Cancer Chemotherapy Handbook,4th ed. St. Louis, Mosby-Year Book, 1993). In some embodiments, thechemotherapeutic agent can be a cytotoxic chemotherapeutic. The term“cytotoxic agent” as used herein refers to a substance that inhibits orprevents the function of cells and/or causes destruction of cells. Theterm is intended to include radioactive isotopes (e.g. At211, I131,I125, Y90, Re186, Re188, Sm153, Bi212, P32 and radioactive isotopes ofLu), chemotherapeutic agents, and toxins, such as small molecule toxinsor enzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably herein to designate a series of amino acid residues,connected to each other by peptide bonds between the alpha-amino andcarboxy groups of adjacent residues. The terms “protein”, and“polypeptide” refer to a polymer of amino acids, including modifiedamino acids (e.g., phosphorylated, glycated, glycosylated, etc.) andamino acid analogs, regardless of its size or function. “Protein” and“polypeptide” are often used in reference to relatively largepolypeptides, whereas the term “peptide” is often used in reference tosmall polypeptides, but usage of these terms in the art overlaps. Theterms “protein” and “polypeptide” are used interchangeably herein whenreferring to a gene product and fragments thereof. Thus, exemplarypolypeptides or proteins include gene products, naturally occurringproteins, homologs, orthologs, paralogs, fragments and otherequivalents, variants, fragments, and analogs of the foregoing.

As used herein an “antibody” refers to IgG, IgM, IgA, IgD or IgEmolecules or antigen-specific antibody fragments thereof (including, butnot limited to, a Fab, F(ab′)₂, Fv, disulphide linked Fv, scFv, singledomain antibody, closed conformation multispecific antibody,disulphide-linked scfv, diabody), whether derived from any species thatnaturally produces an antibody, or created by recombinant DNAtechnology; whether isolated from serum, B-cells, hybridomas,transfectomas, yeast or bacteria.

As described herein, an “antigen” is a molecule that is bound by abinding site on an antibody agent. Typically, antigens are bound byantibody ligands and are capable of raising an antibody response invivo. An antigen can be a polypeptide, protein, nucleic acid or othermolecule or portion thereof. The term “antigenic determinant” refers toan epitope on the antigen recognized by an antigen-binding molecule, andmore particularly, by the antigen-binding site of said molecule.

As used herein, the term “antibody reagent” refers to a polypeptide thatincludes at least one immunoglobulin variable domain or immunoglobulinvariable domain sequence and which specifically binds a given antigen.An antibody reagent can comprise an antibody or a polypeptide comprisingan antigen-binding domain of an antibody. In some embodiments, anantibody reagent can comprise a monoclonal antibody or a polypeptidecomprising an antigen-binding domain of a monoclonal antibody. Forexample, an antibody can include a heavy (H) chain variable region(abbreviated herein as VH), and a light (L) chain variable region(abbreviated herein as VL). In another example, an antibody includes twoheavy (H) chain variable regions and two light (L) chain variableregions. The term “antibody reagent” encompasses antigen-bindingfragments of antibodies (e.g., single chain antibodies, Fab and sFabfragments, F(ab′)2, Fd fragments, Fv fragments, scFv, and domainantibodies (dAb) fragments (see, e.g. de Wildt et al., Eur J. Immunol.1996; 26(3):629-39; which is incorporated by reference herein in itsentirety)) as well as complete antibodies. An antibody can have thestructural features of IgA, IgG, IgE, IgD, IgM (as well as subtypes andcombinations thereof). Antibodies can be from any source, includingmouse, rabbit, pig, rat, and primate (human and non-human primate) andprimatized antibodies. Antibodies also include midibodies, humanizedantibodies, chimeric antibodies, and the like.

The VH and VL regions can be further subdivided into regions ofhypervariability, termed “complementarity determining regions” (“CDR”),interspersed with regions that are more conserved, termed “frameworkregions” (“FR”). The extent of the framework region and CDRS has beenprecisely defined (see, Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242, and Chothia, C.et al. (1987) J. Mol. Biol. 196:901-917; which are incorporated byreference herein in their entireties). Each VH and VL is typicallycomposed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

The terms “antigen-binding fragment” or “antigen-binding domain”, whichare used interchangeably herein are used to refer to one or morefragments of a full length antibody that retain the ability tospecifically bind to a target of interest. Examples of binding fragmentsencompassed within the term “antigen-binding fragment” of a full lengthantibody include (i) a Fab fragment, a monovalent fragment consisting ofthe VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalentfragment including two Fab fragments linked by a disulfide bridge at thehinge region; (iii) an Fd fragment consisting of the VH and CH1 domains;(iv) an Fv fragment consisting of the VL and VH domains of a single armof an antibody, (v) a dAb fragment (Ward et al., (1989) Nature341:544-546; which is incorporated by reference herein in its entirety),which consists of a VH or VL domain; and (vi) an isolatedcomplementarity determining region (CDR) that retains specificantigen-binding functionality. As used herein, the term “specificbinding” refers to a chemical interaction between two molecules,compounds, cells and/or particles wherein the first entity binds to thesecond, target entity with greater specificity and affinity than itbinds to a third entity which is a non-target. In some embodiments,specific binding can refer to an affinity of the first entity for thesecond target entity which is at least 10 times, at least 50 times, atleast 100 times, at least 500 times, at least 1000 times or greater thanthe affinity for the third nontarget entity.

Additionally, and as described herein, a recombinant humanized antibodycan be further optimized to decrease potential immunogenicity, whilemaintaining functional activity, for therapy in humans. In this regard,functional activity means a polypeptide capable of displaying one ormore known functional activities associated with a recombinant antibodyor antibody reagent thereof as described herein. Such functionalactivities include, e.g. the ability to bind to JUP.

As used herein, the term “nucleic acid” or “nucleic acid sequence”refers to any molecule, preferably a polymeric molecule, incorporatingunits of ribonucleic acid, deoxyribonucleic acid or an analog thereof.The nucleic acid can be either single-stranded or double-stranded. Asingle-stranded nucleic acid can be one nucleic acid strand of adenatured double-stranded DNA. Alternatively, it can be asingle-stranded nucleic acid not derived from any double-stranded DNA.In one aspect, the nucleic acid can be DNA. In another aspect, thenucleic acid can be RNA. Suitable nucleic acid molecules are DNA,including genomic DNA or cDNA. Other suitable nucleic acid molecules areRNA, including mRNA.

Aptamers are short synthetic single-stranded oligonucleotides thatspecifically bind to various molecular targets such as small molecules,proteins, nucleic acids, and even cells and tissues. These small nucleicacid molecules can form secondary and tertiary structures capable ofspecifically binding proteins or other cellular targets, and areessentially a chemical equivalent of antibodies. Aptamers are highlyspecific, relatively small in size, and non-immunogenic. Aptamers aregenerally selected from a biopanning method known as SELEX (SystematicEvolution of Ligands by Exponential enrichment) (Ellington et al.Nature. 1990; 346(6287):818-822; Tuerk et al., Science. 1990;249(4968):505-510; Ni et al., Curr Med Chem. 2011; 18(27):4206-14; whichare incorporated by reference herein in their entireties). Methods ofgenerating an apatmer for any given target are well known in the art.Preclinical studies using, e.g. aptamer-siRNA chimeras and aptamertargeted nanoparticle therapeutics have been very successful in mousemodels of cancer and HIV (Ni et al., Curr Med Chem. 2011;18(27):4206-14).

Inhibitors of the expression of a given gene can be an inhibitorynucleic acid. In some embodiments, the inhibitory nucleic acid is aninhibitory RNA (iRNA). Double-stranded RNA molecules (dsRNA) have beenshown to block gene expression in a highly conserved regulatorymechanism known as RNA interference (RNAi). The inhibitory nucleic acidsdescribed herein can include an RNA strand (the antisense strand) havinga region which is 30 nucleotides or less in length, i.e., 15-30nucleotides in length, generally 19-24 nucleotides in length, whichregion is substantially complementary to at least part of the targetedmRNA transcript. The use of these iRNAs enables the targeted degradationof mRNA transcripts, resulting in decreased expression and/or activityof the target.

As used herein, the term “iRNA” refers to an agent that contains RNA asthat term is defined herein, and which mediates the targeted cleavage ofan RNA transcript via an RNA-induced silencing complex (RISC) pathway.In one embodiment, an iRNA as described herein effects inhibition of theexpression and/or activity of JUP. In certain embodiments, contacting acell with the inhibitor (e.g. an iRNA) results in a decrease in thetarget mRNA level in a cell by at least about 5%, about 10%, about 20%,about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about90%, about 95%, about 99%, up to and including 100% of the target mRNAlevel found in the cell without the presence of the iRNA.

In some embodiments, the iRNA can be a dsRNA. A dsRNA includes two RNAstrands that are sufficiently complementary to hybridize to form aduplex structure under conditions in which the dsRNA will be used. Onestrand of a dsRNA (the antisense strand) includes a region ofcomplementarity that is substantially complementary, and generally fullycomplementary, to a target sequence. The target sequence can be derivedfrom the sequence of an mRNA formed during the expression of the target.The other strand (the sense strand) includes a region that iscomplementary to the antisense strand, such that the two strandshybridize and form a duplex structure when combined under suitableconditions. Generally, the duplex structure is between 15 and 30inclusive, more generally between 18 and 25 inclusive, yet moregenerally between 19 and 24 inclusive, and most generally between 19 and21 base pairs in length, inclusive. Similarly, the region ofcomplementarity to the target sequence is between 15 and 30 inclusive,more generally between 18 and 25 inclusive, yet more generally between19 and 24 inclusive, and most generally between 19 and 21 nucleotides inlength, inclusive. In some embodiments, the dsRNA is between 15 and 20nucleotides in length, inclusive, and in other embodiments, the dsRNA isbetween 25 and 30 nucleotides in length, inclusive. As the ordinarilyskilled person will recognize, the targeted region of an RNA targetedfor cleavage will most often be part of a larger RNA molecule, often anmRNA molecule. Where relevant, a “part” of an mRNA target is acontiguous sequence of an mRNA target of sufficient length to be asubstrate for RNAi-directed cleavage (i.e., cleavage through a RISCpathway). dsRNAs having duplexes as short as 9 base pairs can, undersome circumstances, mediate RNAi-directed RNA cleavage. Most often atarget will be at least 15 nucleotides in length, preferably 15-30nucleotides in length.

In yet another embodiment, the RNA of an iRNA, e.g., a dsRNA, ischemically modified to enhance stability or other beneficialcharacteristics. The nucleic acids featured in the invention may besynthesized and/or modified by methods well established in the art, suchas those described in “Current protocols in nucleic acid chemistry,”Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y.,USA, which is hereby incorporated herein by reference. Modificationsinclude, for example, (a) end modifications, e.g., 5′ end modifications(phosphorylation, conjugation, inverted linkages, etc.) 3′ endmodifications (conjugation, DNA nucleotides, inverted linkages, etc.),(b) base modifications, e.g., replacement with stabilizing bases,destabilizing bases, or bases that base pair with an expanded repertoireof partners, removal of bases (abasic nucleotides), or conjugated bases,(c) sugar modifications (e.g., at the 2′ position or 4′ position) orreplacement of the sugar, as well as (d) backbone modifications,including modification or replacement of the phosphodiester linkages.Specific examples of RNA compounds useful in the embodiments describedherein include, but are not limited to RNAs containing modifiedbackbones or no natural internucleoside linkages. RNAs having modifiedbackbones include, among others, those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified RNAs that do not have aphosphorus atom in their internucleoside backbone can also be consideredto be oligonucleosides. In particular embodiments, the modified RNA willhave a phosphorus atom in its internucleoside backbone.

Modified RNA backbones can include, for example, phosphorothioates,chiral phosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those) having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included. RepresentativeU.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170;6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423;6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294;6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat.RE39464, each of which is herein incorporated by reference

Modified RNA backbones that do not include a phosphorus atom thereinhave backbones that are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatoms and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts. Representative U.S. patents that teach thepreparation of the above oligonucleosides include, but are not limitedto, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134;5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257;5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and, 5,677,439, each of which is hereinincorporated by reference.

In other RNA mimetics suitable or contemplated for use in iRNAs, boththe sugar and the internucleoside linkage, i.e., the backbone, of thenucleotide units are replaced with novel groups. The base units aremaintained for hybridization with an appropriate nucleic acid targetcompound. One such oligomeric compound, an RNA mimetic that has beenshown to have excellent hybridization properties, is referred to as apeptide nucleic acid (PNA). In PNA compounds, the sugar backbone of anRNA is replaced with an amide containing backbone, in particular anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative U.S. patents that teach the preparation of PNAcompounds include, but are not limited to, U.S. Pat. Nos. 5,539,082;5,714,331; and 5,719,262, each of which is herein incorporated byreference. Further teaching of PNA compounds can be found, for example,in Nielsen et al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the invention include RNAs withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂-[known as amethylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂—[wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAsfeatured herein have morpholino backbone structures of theabove-referenced U.S. Pat. No. 5,034,506.

Modified RNAs can also contain one or more substituted sugar moieties.The iRNAs, e.g., dsRNAs, featured herein can include one of thefollowing at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-Co-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(.n)OCH₃, O(CH₂)_(n)NH₂,O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where nand m are from 1 to about 10. In other embodiments, dsRNAs include oneof the following at the 2′ position: C₁ to C₁₀ lower alkyl, substitutedlower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN,Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of aniRNA, or a group for improving the pharmacodynamic properties of aniRNA, and other substituents having similar properties. In someembodiments, the modification includes a 2′-methoxyethoxy(2′-O-CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-M0E) (Martinet al., Helv. Chico. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxygroup. Another exemplary modification is 2′-dimethylaminooxyethoxy,i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described inexamples herein below, and 2′-dimethylaminoethoxyethoxy (also known inthe art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples herein below.

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications can alsobe made at other positions on the RNA of an iRNA, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkeddsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs may alsohave sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application, and each of which is hereinincorporated by reference.

An iRNA can also include nucleobase (often referred to in the art simplyas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C) anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substitutedadenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in Modified Nucleosides in Biochemistry, Biotechnology andMedicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in TheConcise Encyclopedia Of Polymer Science And Engineering, pages 858-859,Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed byEnglisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Researchand Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRCPress, 1993. Certain of these nucleobases are particularly useful forincreasing the binding affinity of the oligomeric compounds featured inthe invention. These include 5-substituted pyrimidines, 6-azapyrimidinesand N-2, N-6 and 0-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., Eds., dsRNA Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are exemplary base substitutions, evenmore particularly when combined with 2′-O-methoxyethyl sugarmodifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. No.3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;5,614,617; 5,681,941; 6,015,886; 6,147,200; 6,166,197; 6,222,025;6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610;7,427,672; and 7,495,088, each of which is herein incorporated byreference, and U.S. Pat. No. 5,750,692, also herein incorporated byreference.

The RNA of an iRNA can also be modified to include one or more lockednucleic acids (LNA). A locked nucleic acid is a nucleotide having amodified ribose moiety in which the ribose moiety comprises an extrabridge connecting the 2′ and 4′ carbons. This structure effectively“locks” the ribose in the 3′-endo structural conformation. The additionof locked nucleic acids to siRNAs has been shown to increase siRNAstability in serum, and to reduce off-target effects (Elmen, J. et al.,(2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007)Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic AcidsResearch 31(12):3185-3193). Representative U.S. Patents that teach thepreparation of locked nucleic acid nucleotides include, but are notlimited to, the following: U.S. Pat. Nos. 6,268,490; 6,670,461;6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845, each of whichis herein incorporated by reference in its entirety.

Another modification of the RNA of an iRNA featured in the inventioninvolves chemically linking to the RNA one or more ligands, moieties orconjugates that enhance the activity, cellular distribution,pharmacokinetic properties, or cellular uptake of the iRNA. Suchmoieties include but are not limited to lipid moieties such as acholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989,86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let.,1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan etal., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg.Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser etal., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g.,dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991,10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuket al., Biochimie, 1993, 75:49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res.,1990, 18:3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264:229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923-937).

As used herein, the terms “treat,” “treatment,” “treating,” or“amelioration” refer to therapeutic treatments, wherein the object is toreverse, alleviate, ameliorate, inhibit, slow down or stop theprogression or severity of a condition associated with a disease ordisorder, e.g. cancer. The term “treating” includes reducing oralleviating at least one adverse effect or symptom of a condition,disease or disorder associated with a cancer. Treatment is generally“effective” if one or more symptoms or clinical markers are reduced.Alternatively, treatment is “effective” if the progression of a diseaseis reduced or halted. That is, “treatment” includes not just theimprovement of symptoms or markers, but also a cessation of, or at leastslowing of, progress or worsening of symptoms compared to what would beexpected in the absence of treatment. Beneficial or desired clinicalresults include, but are not limited to, alleviation of one or moresymptom(s), diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, remission (whetherpartial or total), and/or decreased mortality, whether detectable orundetectable. The term “treatment” of a disease also includes providingrelief from the symptoms or side-effects of the disease (includingpalliative treatment).

As used herein, the term “pharmaceutical composition” refers to theactive agent in combination with a pharmaceutically acceptable carriere.g. a carrier commonly used in the pharmaceutical industry. The phrase“pharmaceutically acceptable” is employed herein to refer to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used herein, the term “administering,” refers to the placement of acompound as disclosed herein into a subject by a method or route whichresults in at least partial delivery of the agent at a desired site.Pharmaceutical compositions comprising the compounds disclosed hereincan be administered by any appropriate route which results in aneffective treatment in the subject.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) or greater difference.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean±1%.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the method or composition, yet open to the inclusion ofunspecified elements, whether essential or not.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

Definitions of common terms in cell biology and molecular biology can befound in “The Merck Manual of Diagnosis and Therapy”, 19th Edition,published by Merck Research Laboratories, 2006 (ISBN 0-911910-19-0);Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology,published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); BenjaminLewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN-10:0763766321); Kendrew et al. (eds.), Molecular Biology and Biotechnology:a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995(ISBN 1-56081-569-8) and Current Protocols in Protein Sciences 2009,Wiley Intersciences, Coligan et al., eds.

Unless otherwise stated, the present invention was performed usingstandard procedures, as described, for example in Sambrook et al.,Molecular Cloning: A Laboratory Manual (4 ed.), Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., USA (2012); Davis et al.,Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc.,New York, USA (1995); or Methods in Enzymology: Guide to MolecularCloning Techniques Vol. 152, S. L. Berger and A. R. Kimmel Eds.,Academic Press Inc., San Diego, USA (1987); Current Protocols in ProteinScience (CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons,Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et.al. ed., John Wiley and Sons, Inc.), and Culture of Animal Cells: AManual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5thedition (2005), Animal Cell Culture Methods (Methods in Cell Biology,Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, 1stedition, 1998) which are all incorporated by reference herein in theirentireties.

Other terms are defined herein within the description of the variousaspects of the invention.

All patents and other publications; including literature references,issued patents, published patent applications, and co-pending patentapplications; cited throughout this application are expresslyincorporated herein by reference for the purpose of describing anddisclosing, for example, the methodologies described in suchpublications that might be used in connection with the technologydescribed herein. These publications are provided solely for theirdisclosure prior to the filing date of the present application. Nothingin this regard should be construed as an admission that the inventorsare not entitled to antedate such disclosure by virtue of priorinvention or for any other reason. All statements as to the date orrepresentation as to the contents of these documents is based on theinformation available to the applicants and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. For example, while methodsteps or functions are presented in a given order, alternativeembodiments may perform functions in a different order, or functions maybe performed substantially concurrently. The teachings of the disclosureprovided herein can be applied to other procedures or methods asappropriate. The various embodiments described herein can be combined toprovide further embodiments. Aspects of the disclosure can be modified,if necessary, to employ the compositions, functions and concepts of theabove references and application to provide yet further embodiments ofthe disclosure. Moreover, due to biological functional equivalencyconsiderations, some changes can be made in protein structure withoutaffecting the biological or chemical action in kind or amount. These andother changes can be made to the disclosure in light of the detaileddescription. All such modifications are intended to be included withinthe scope of the appended claims.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure.

The technology described herein is further illustrated by the followingexamples which in no way should be construed as being further limiting.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

-   -   1. A method of treating cancer, the method comprising measuring        the level of circulating tumor cell (CTC) clusters in a sample        obtained from a subject with a cancer;        -   administering a treatment to prevent or reduce metastasis if            the level of CTC clusters is increased relative to a control            level; and        -   not administering a treatment to prevent or reduce            metastasis if the level of CTC clusters is not increased            relative to a control level.    -   2. A method of treating cancer, the method comprising        administering a treatment to prevent or reduce metastasis in a        subject determined to have a level of CTC cluster which is        increased relative to a control level.    -   3. The method of paragraph 2, the method further comprising not        administering a treatment to prevent or reduce metastasis in a        subject determined to have a level of CTC clusters is not        increased relative to a control level.    -   4. The method of any of paragraphs 1-3, wherein the cancer is a        breast or epithelial cancer.    -   5. The method of any of paragraphs 1-4, wherein the treatment to        prevent or reduce metastasis is selected from the group        consisting of:        -   a method of any of paragraphs 15-19; chemotherapy; radiation            therapy; or removal of a tumor.    -   6. The method of any of paragraphs 1-5, wherein not        administering a treatment can comprise a clinical approach of        monitoring without therapeutic intervention.    -   7. The method of any of paragraphs 1-6, wherein the level of CTC        clusters is measured by measuring the expression level of a CTC        cluster (CTC-C) marker gene in the sample obtained from the        subject;        -   wherein the CTC-C marker gene is a gene selected from the            list of Table 2, 3, or 4.    -   8. The method of paragraph 7, wherein the CTC-C marker gene is        plakoglobin.    -   9. The method of any of paragraphs 7-8, wherein the expression        level of a CTC-C marker gene in circulating tumor cells in the        sample is measured.    -   10. The method of any of paragraphs 7-9, wherein the expression        level of a CTC-C marker gene in cancer cells obtained from the        subject is measured.    -   11. The method of any of paragraphs 1-10, wherein the level of        CTC clusters is measured using a ^(HB)CTC-Chip.    -   12. The method of any of paragraphs 1-11, wherein the subject is        a subject in need of treatment for cancer.    -   13. The method of any of paragraphs 1-12, wherein an increased        level of CTC clusters is a level at least 1.5× greater than the        control level.    -   14. The method of any of paragraphs 1-13, wherein an increased        level of plakoglobin expression is a level at least 1.5× greater        than the control level.    -   15. A method of treating cancer metastasis, the method        comprising reducing the level of expression or activity of a        CTC-C marker gene; wherein the CTC-C marker gene is a gene        selected from the list of Table 2, 3, or 4.    -   16. The method of paragraph 15, wherein reducing the level of        expression or activity of a CTC-C marker gene comprises        administering a CTC-C marker gene inhibitory nucleic acid.    -   17. The method of paragraph 16, wherein the inhibitory nucleic        acid is a siRNA.    -   18. The method of any of paragraphs 15-17, wherein the CTC-C        marker gene is plakoglobin.    -   19. The method of any of paragraphs 15-18, wherein the cancer is        a breast or epithelial cancer.    -   20. An assay comprising:        -   measuring the level of circulating tumor cell (CTC) clusters            in a sample obtained from a subject with cancer;        -   determining the subject to be at increased risk of            metastasis of the cancer if the level of CTC clusters is            increased relative to a control level.    -   21. The assay of paragraph 20, wherein the cancer is a breast or        epithelial cancer.    -   22. The assay of any of paragraphs 20-21, wherein the level of        CTC clusters is measured by measuring the expression level of a        CTC cluster (CTC-C) marker gene in the sample obtained from the        subject;        -   wherein the CTC-C marker gene is a gene selected from the            list of Table 2, 3 or 4.    -   23. The assay of paragraph 22, wherein the CTC-C marker gene is        plakoglobin.    -   24. The assay of any of paragraphs 20-23, wherein the expression        level of a CTC-C marker gene in circulating tumor cells in the        sample is measured.    -   25. The assay of any of paragraphs 20-24, wherein the expression        level of a CTC-C marker gene in cancer cells obtained from the        subject is measured.    -   26. The assay of any of paragraphs 20-25, wherein the level of        CTC clusters is measured using a ^(HB)CTC-Chip.    -   27. The assay of any of paragraphs 20-26, wherein the subject is        a subject in need of treatment for cancer.    -   28. The assay of any of paragraphs 20-27, wherein an increased        level of CTC clusters is a level at least 1.5× greater than the        control level.    -   29. The assay of any of paragraphs 20-28, wherein an increased        level of plakoglobin expression is a level at least 1.5× greater        than the control level.    -   30. A method of determining if a subject is at increased risk of        metastasis, the method comprising:        -   measuring the level of circulating tumor cell (CTC) clusters            in a sample obtained from a subject with a cancer;        -   determining the subject to be at increased risk of            metastasis of the cancer if the level of CTC clusters is            increased relative to a control level.    -   31. The method of paragraph 30, wherein the cancer is a breast        or epithelial cancer.    -   32. The method of any of paragraphs 30-31, wherein the level of        CTC clusters is measured by measuring the expression level of a        CTC cluster (CTC-C) marker gene in the sample obtained from the        subject;        -   wherein the CTC-C marker gene is a gene selected from the            list of Table 2, 3, or 4.    -   33. The method of paragraph 32, wherein the CTC-C marker gene is        plakoglobin.    -   34. The method of any of paragraphs 30-33, wherein the        expression level of a CTC-C marker gene in circulating tumor        cells in the sample is measured.    -   35. The method of any of paragraphs 30-34, wherein the        expression level of a CTC-C marker gene in cancer cells obtained        from the subject is measured.    -   36. The method of any of paragraphs 30-35, wherein the level of        CTC clusters is measured using a ^(HB)CTC-Chip.    -   37. The method of any of paragraphs 30-36, wherein the subject        is a subject in need of treatment for cancer.    -   38. The method of any of paragraphs 30-37, wherein an increased        level of CTC clusters is a level at least 1.5× greater than the        control level.    -   39. The method of any of paragraphs 30-38, wherein an increased        level of plakoglobin expression is a level at least 1.5× greater        than the control level.    -   40. A method of reducing the level of circulating tumor cell        (CTC) clusters in a subject with cancer, the method comprising        reducing the level of expression or activity of a CTC-C marker        gene; wherein the CTC-C marker gene is a gene selected from the        list of Table 2, 3, or 4.    -   41. The method of paragraph 40, wherein reducing the level of        expression or activity of a CTC-C marker gene comprises        administering a CTC-C marker gene inhibitory nucleic acid.    -   42. The method of paragraph 41, wherein the inhibitory nucleic        acid is a siRNA.    -   43. The method of any of paragraphs 40-42, wherein the CTC-C        marker gene is plakoglobin.    -   44. The method of any of paragraphs 40-43, wherein the cancer is        a breast or epithelial cancer.

EXAMPLES Example 1: Circulating Tumor Cells Clusters are OligoclonalPrecursors of Breast Cancer Metastasis

Clusters of circulating tumor cells (CTC-clusters) have been observed inpatients with epithelial cancers but their role in the metastaticprocess has remained elusive. It is demonstrated herein that thepresence of CTC-clusters in breast cancer patients directly correlateswith increased metastatic progression. In mouse models, the metastaticpotential of CTC-clusters and that of single CTCs was assessed, and itwas determined that CTC-clusters are 48.9 times more metastatic thansingle CTCs. Moreover, adopting intravital imaging and in vivo flowcytometry, it was determined that CTC-clusters are oligoclonal unitsderived from the primary tumor, and that they are associated to a fasterclearance rate from the bloodstream than single CTCs. RNA sequencing ofmatched CTC-clusters versus single CTCs in breast cancer patientsallowed the identification of CTC-clusters-associated transcripts. Amongthese, the cell-to-cell junction protein Plakoglobin was found to berequired for CTC-clusters formation and breast cancer metastasis.Accordingly, high Plakoglobin expression in the primary tumor isassociated with a diminished metastasis-free survival of patients. Insummary, the data described herein indicate that CTC-clusters areoligoclonal metastatic precursors, and that their disruption viaPlakoglobin inhibition decreases the metastatic spread of breast cancer.

INTRODUCTION

Breast cancer is a heterogeneous disease that culminates with metastasisto vital organs such as bone, lung, liver and brain, and generally,metastasis accounts for the vast majority of cancer-related deaths (1).The current model of blood-borne metastasis is based on sequential stepsstarting from intravasation of single primary tumor cells into thebloodstream, survival in the circulation, extravasation and colonizationto distant sites (2). Circulating tumor cells (CTCs) have been detectedin the majority of epithelial cancers (3) and they hold the key tounderstanding early dissemination events in cancer. Interestingly, thenumber of CTCs largely exceeds the number of metastatic lesions inpatients, indicating a cellular hierarchy among CTCs and indicating thatthe majority of CTCs is likely to die in the bloodstream, with only aminor fraction representing viable metastatic precursors. Theidentification of the metastatic pool within breast CTCs and itsmolecular characterization has the potential to refine our understandingof breast cancer metastasis and can permit the development of new agentsfor the treatment of metastatic human tumors.

A recent and surprising observation by the inventors was theidentification of clusters of CTCs (CTC-clusters) in the circulation ofpatients with cancer (4, 5). It is demonstrated herein, using acombination of breast cancer patient samples, mouse models andnext-generation sequencing, that breast CTC-clusters highly representthe metastatic precursor population within breast CTCs, and thatdisruption of CTC-clusters is associated with diminished metastaticspread.

Results

The Presence of CTC-clusters in Patients Correlates with MetastaticProgression. The role of CTC-clusters in breast cancer metastasis anddisease progression has not been previously investigated. Themicrofluidic herringbone (HB)-CTC-chip (4) was used herein to captureand enumerate single CTCs and CTC-clusters from the blood of 79 breastcancer patients during multiple time points (corresponding to a total of265 data points). Capture of breast CTCs was achieved with an antibodycocktail directed against EpCAM, epithelial growth factor receptor(EGFR) and human epithelial growth factor receptor 2 (HER2). The chipwas then stained with anti-wide spectrum Cytokeratin (CK) to identifyCTCs and anti-CD45 to assess white blood cells (WBCs) contamination(data not shown). First, CTCs were identified in 54 out of 79 patients(FIG. 1A). Second, it was observed that seven of the 54 CTCs-positivepatients were characterized by the presence of high counts ofCTC-clusters across multiple time points (FIG. 1A). The remaining 47patients were characterized by either the presence of CTC-clusters atonly one time point or by the complete absence of CTC-clusters acrossall time points (FIG. 1A).

Progression-free survival (PFS) data was obtained for the patients whosedisease progressed during the time frame that was considered for CTCsenumeration (Table 1). It was found that CTC-clusters-enriched patientsprogressed to metastasis more rapidly than the single CTCs-enriched ones(mean progression-free survival time was 76.1 days forCTC-cluster-enriched and 160.6 days for single CTCs-enriched patients)(FIG. 1B and (Table 1). These results indicate that the presence ofCTC-clusters is associated with faster metastatic spread and diseaseprogression in breast cancer patients.

CTC-clusters harbor an increased metastatic potential compared to singleCTCs. Using breast cancer mouse models, the metastatic potential of bothsingle CTCs and CTC-clusters was dissected and quantified. To this end,an in vitro assay was developed that allowed the generation of a singlecell suspension as well as a suspension of clustered cells (ranging from2 to −30 cell clusters) starting from a monolayer culture of thelung-metastatic variant of MDA-MB-231 (LM2) cells labeled withGFP-Luciferase (6). Secondly, 200,000 LM2 cells prepared as single cells(LM2-SCs) or as clusters (LM2-CLs) were injected in the tail vein ofimmunodeficient mice. Interestingly, both LM2-SCs and LM2-CLs reachedthe lungs with equal efficiency (day 0) (data not shown). However, whileLM2-SCs underwent massive apoptosis in the lungs upon injection, LM2-CLsshowed significantly higher resistance to apoptosis and faster growthrate (data not shown and FIGS. 2A and 6A). In addition, mice injectedwith LM2-CLs had a reduced overall survival (mean survival time LM2-CLsmice=12.7 weeks vs LM2-SCs mice=15.7 weeks) (FIG. 6B).

In a more clinically relevant setting, orthotopic xenograft models wereused to quantify the metastatic potential of tumor-derived breastCTC-clusters and single CTCs. First, LM2 cells were engineered toexpress either green fluorescent protein (LM2-GFP) or mCherry(LM2-mCherry). LM2-GFP and LM2-mCherry cells were then mixed 1:1 andinjected in the mammary fat pad of immunodeficient mice. It was reasonedthat, upon tumor development, single CTCs as well as single CTCs-derivedmetastases would be either GFP- or mCherry-positive. In contrast,CTC-clusters as well as CTC-clusters-derived metastases would be formedby both GFP- and mCherry-positive LM2 cells. Five weeks upon primarybreast tumor formation, it was confirmed by immunohistochemical (IHC)staining that LM2-GFP and LM2-mCherry cells retained a 1:1 distributionin the primary tumor site (data not shown). Single CTCs and CTC-clusterswere captured and quantified with a HB-CTC-chip functionalized withanti-EpCAM and anti-EGFR antibodies. At the same time, the mouse lungswere stained with anti-GFP and anti-mCherry antibodies and the number ofGFP- or mCherry-positive foci (derived from a single CTC) as well as thenumber of GFP- and mCherry-positive foci (derived from a CTC-cluster)were quantified (data not shown). A mean of 2,486 CTCs per mouse wereobserved, of which 2.4% were multicolor CTC-clusters (FIG. 2B).Strikingly, a mean of 323 lung foci per mouse were counted, of which52.9% were CTC-clusters derived (FIG. 2B). The lung metastasis data wasnormalized with the number of single CTCs and CTC-clusters, and it wasconcluded that CTC-clusters are 48.9 times more metastatic than singleCTCs in the LM2 xenograft model (FIG. 2C).

CTC-clusters are primary tumor-derived oligoclonal units associated withhigh clearance rate from the bloodstream. The multicolor nature ofCTC-clusters in the mouse orthotopic xenograft model indicates thatCTC-clusters are heterogeneous units and do not derive from a singleproliferating CTC (data not shown). However, to rule out that a)CTC-clusters are formed in circulation from the aggregation of multiplesingle CTCs and that b) CTC-clusters are an artifact of the HB-CTC-chip,intravital imaging of the primary tumor site in mice injected with a 1:1mixture of LM2-GFP and LM2-mCherry cells was performed (as describedabove). Five weeks after orthotopic injection, draining vessels adjacentto the primary tumor mass were imaged, where early intravasation eventscan be observed and where the possibility for single CTCs to aggregatein circulation is lowest due to the very short distance between theintravasation site and focal plane. Strikingly, numerous events of earlyintravasation of multicolor CTC-clusters in living animals as well asmultiple single CTCs were detected (data not shown).

Next, given their oligoclonal nature, it was reasoned that CTC-clusterscould be more likely—than single CTCs—to be trapped in small capillaries(e.g. those in the lungs), therefore associated with a faster clearancerate from the bloodstream. In vivo flow cytometry (IVFC) was used tomeasure clearance rates of LM2-SCs or LM2-CLs injected in the tail veinof immunodeficient mice. Particularly, circulating DiD-labeled LM2-SCsor LM2-CLs were detected real time in the ear blood vessels for a totalof 55 minutes in each mouse. It was found that LM2-CLs cleared morerapidly than LM-SCs, suggesting that aggregated cells are more likely tobe trapped in small capillaries (FIG. 3). All together, these resultsindicate that CTC-clusters are oligoclonal units derived from theprimary tumor and that they are associated with a faster clearance ratefrom the bloodstream and propensity to get trapped in small capillaries.

RNA sequencing of matched CTC-clusters and single CTCs from breastcancer patients reveals CTC-clusters-associated genes. Given the findingdescribed herein, that CTC-clusters retain a higher metastatic potentialthan single CTCs, it was asked if a specific gene set was highlyexpressed in CTC-clusters compared to matched single CTCs from the samepatient. To this end, blood specimens were collected from 10 breastcancer patients with metastatic disease and a CTCs-enriched productderived with the inventors' recently developed antigen-independent^(neg)CTC-iChip (7). Particularly, the ^(neg)CTC-iChip allows bloodsamples to be depleted of red blood cells (RBCs), platelets and plasmaproteins by hydrodynamic size-based sorting and subsequently ofleukocytes by immunomagnetical targeting of both CD45 and CD66b antigens(7). A live staining was then performed on the resulting CTC-enrichedsolution with a) CTCs-directed antibodies anti-EpCAM, anti-EGFR,anti-HER2, anti-CDH11 and anti-MET conjugated with an Alexa488fluorophore and b) white blood cells (WBCs)-directed antibodiesanti-CD45, anti-CD14 and anti-CD16 conjugated with a TexasRedfluorophore (FIG. 4A). This procedure allowed the identification ofsingle CTCs and CTC-clusters (labeled in green), versus contaminant WBCs(labeled in red) and red blood cells (RBCs, unlabeled) (data not shown).With a micromanipulator single CTCs versus CTC-clusters wereindividually isolated for each sample, and CTCs-derived RNA wassubjected to RNA sequencing. Normalized expression profiles were derivedfor a total of 29 samples (15 pools of single CTCs and 14 CTC-clusters)derived from 10 breast cancer patients. Samples that showed highexpression of contaminant WBC markers at the RNA level were excludedfrom the analysis. Unsupervised hierarchical clustering showed noobvious distinction at the global gene expression level between singleCTCs and CTC-cluster samples (FIGS. 4B and 7), indicating thatinter-patient differences were prevailing the intra-patient geneexpression changes between CTC-clusters and single CTCs.

For each patient, gene expression data of each CTC-cluster versus eachsingle-CTC sample was compared to generate a list of 31CTC-clusters-associated genes (q<0.01, log 2FC>1 in more than 70%intra-patient comparisons) (FIGS. 4C and 9). Among the top upregulatedtranscripts in CTC-clusters was Plakoglobin (JUP) (FIGS. 4C, 4D, and 9),a member of the Armadillo family of proteins and an important componentof desmosomes and adherence junctions (8-10). Previously, Plakoglobinhas been shown to play both positive and negative roles in malignancies.For example, Plakoglobin overexpression in transformed rat kidneyepithelial cells promotes unregulated growth, foci formation and c-Mycactivation (11). In human squamous carcinoma cells, Plakoglobin wasshown to cause inhibition of apoptosis, unregulated growth and fociformation via activation of the pro-survival gene Bcl-2 (12).Accordingly, Plakoglobin mutations have been reported in hormonerefractory prostate cancers, and these coincide with accumulation ofnuclear Plakoglobin and increase in Bcl-2 expression (13). However, lossof heterozygosity and hypermethylation of the Plakoglobin promoter havebeen reported in localized prostate cancer (13). Plakoglobin expressionwas confirmed at the protein level in breast CTC-clusters derived from apatient with metastatic disease (data not shown). These results showthat, when compared to single CTCs, breast CTC-clusters arecharacterized by the upregulation of a subset of genes and thatcell-cell junction proteins such as Plakoglobin are involved in theirformation and maintenance.

Plakoglobin is required for CTC-clusters formation and breast cancermetastasis. Plakoglobin expression levels were assessed in a primarybreast tumor and bone metastasis biopsies of a breast cancer patientmatched with high CTC-clusters counts. Particularly, Plakoglobinstaining was combined with the endothelial cells marker CD31 toinvestigate whether high Plakoglobin expression occurred in proximity toblood vessels. Primary tumor cells were found to express Plakoglobin atdifferent levels, with both “high Plakoglobin” and “low Plakoglobin”regions being localized next to blood vessels (data not shown).Consistently, both “high Plakoglobin”- and “low Plakoglobin”-expressingcells were observed in the metastatic foci (data not shown), indicatingthat CTC-clusters are likely arise from “high Plakoglobin” regions inthe primary tumor, express high levels of Plakoglobin while circulating,and retain its expression in the metastatic site.

The requirement of Plakoglobin for cell-to-cell adhesion in a panel ofnon-transformed human mammary epithelial cells (HMEC and MCF10A) andhuman breast cancer cells (MDA-MB-231-LM2, BT474, MCF7, T47D, BT549,BT20 and ZR-75-1) was measured. Stable Plakoglobin knockdown wasachieved by lentiviral transduction of Plakoglobin shRNAs (Plakoglobinsh1 and sh2) in all cell lines (FIG. 8A). A Vybrant™ cell-to-celladhesion assay was performed and it was observed that Plakoglobinrequirement for cell-to-cell adhesion was higher for breast cancer cellscompared to normal breast cells (FIG. 5A). Moreover, upon Plakoglobinknockdown in monolayer cultures, disruption of cellular colonies wasobserved in breast cancer cells but not in non-transformed mammaryepithelial cells (data not shown), supporting the hypothesis that thatnon-neoplastic cells may rely on additional/different subsets of genesfor cell-to-cell adhesion mechanisms (14, 15).

LM2-Luciferase cells expressing a control or Plakoglobin shRNAs wereinjected in the mammary fat pad of immunodeficient mice and tumor growthas well as CTCs abundance was measured. Plakoglobin knockdown for 30days did not alter the tumor growth rate (FIGS. 5B and 8B) or the totalnumber of single CTCs derived from the primary tumor (FIG. 5C). However,the number of tumor-derived CTC-clusters was significantly reduced inmice bearing LM2-Plakoglobin shRNAs tumors compared to control mice(FIGS. 5C and 8B). In parallel, bioluminescence imaging of mouse lungswas performed and it was observed that Plakoglobin knockdown reduced themetastatic capacity of LM2 tumors. Particularly, an approximate 80%reduction of the metastatic lung burden in mice bearing LM2-PlakoglobinshRNAs tumors was observed (FIG. 5D). Finally, given the observationsthat Plakoglobin levels are likely to play an important role in theformation of CTC-clusters and metastasis, distant metastasis-freesurvival was assessed in a cohort of 1353 breast cancer patientsclassified into “low Plakoglobin” and “high Plakoglobin”, according toPlakoglobin expression levels in their primary tumor. It was found thatthe patients whose primary tumor expressed high levels of Plakoglobinwere associated to faster disease progression than the “low Plakoglobin”counterpart (FIG. 5E).

These data indicate that “high Plakoglobin” regions within the primarytumor are likely to shed oligoclonal CTC-clusters in the bloodstream(FIG. 5F). Accordingly, disruption of tumor-derived CTC-clusters viaPlakoglobin depletion reduces the metastatic burden in breast canceranimal models. In summary, it is demonstrated herein that CTC-clustersrepresent a highly metastatic population within CTCs and that targetingCTC-clusters may be effective to reduce the metastatic spread of breastcancer.

DISCUSSION

While CTC-clusters have been observed in patients with cancers ofdifferent origin (4, 16), their contribution to the metastatic processwas not previously investigated. In this study, it is demonstrated thatthe presence of breast CTC-clusters in patients correlates with areduced progression-free survival, and that CTC-clusters represent ahighly metastatic population within CTCs. Moreover, CTC-clusters appearto be of oligoclonal nature and to originate from regions of the primarytumor characterized by high expression of Plakoglobin, a cell-to-celljunction mediator. In animal models, Plakoglobin knockdown in theprimary tumor decreases the number of tumor-derived CTC-clusters as wellas lung metastasis.

The RNA sequencing data described herein has led to the identificationof a set of CTC-cluster-associated transcripts including Plakoglobin.The contribution of Plakoglobin to CTC-clusters formation was clarified.

The oligoclonal nature of breast CTC-clusters highlights that, inaddition to an “active” metastatic mechanism of single motile CTCs, a“passive” metastatic program is likely to be relevant in patients withbreast cancer. Areas within the primary tumor with high expression ofcell-to-cell junction proteins such as Plakoglobin may increase thelikelihood of tumor emboli to intravasate into the bloodstream. As such,CTC-clusters a) retain high Plakoglobin expression while circulating, b)are more likely to be trapped in small capillaries and clear from thecirculation with a faster rate than single CTCs and c) resist better tothe apoptotic stress induced by a foreign environment such a s lungs,bone, liver and/or other metastatic sites.

Described herein is evidence that CTC-clusters are oligoclonalmetastatic precursors in breast cancer, and that disruption ofCTC-clusters via Plakoglobin knockdown decreases the metastatic spreadof breast cancer. The clinical relevance of CTC-clusters and theirsignaling mechanisms sustaining cell-to-cell junctions and metastaticpotential permits novel therapeutic targets for the treatment ofmetastatic breast tumors.

Methods

Circulating tumor cells capture and identification. Blood specimens forCTCs analysis were obtained after informed patient consent, per IRBprotocol (05-300), at the Massachusetts General Hospital. A maximum of20 ml of blood was drawn in EDTA vacutainers. Within four hours from theblood draw, approximately 3 ml of blood was processed through theHB-CTC-Chip or 6-12 ml of blood was processed through the CTC-iChip. Formouse studies, the blood was retrieved via cardiac puncture andapproximately 1 ml of blood was processed through the HB-CTC-Chip.

HB-CTC-Chips were manufactured on site at the Massachusetts GeneralHospital Cancer Center/BioMEMS Resource Facility. Chips werefunctionalized as previously described (5) with a cocktail of 10 μg/mleach of biotinylated antibodies anti-EpCAM (R&D Systems), anti-EGFR(Cetuximab, Lilly) and anti-HER2 (R&D Systems). Captured cells on theHB-CTC-Chip were fixed with 4% paraformaldehyde and washed with PBS.Fixed cells were then permeabilized with 1% NP40 in PBS, blocked with 3%goat serum/2% BSA, and immunostained with anti-wide spectrum cytokeratin(Abcam), anti-CD-45 (Abcam), anti-Plakoglobin (Sigma Aldrich) and DAPI.Alternatively, GFP- or mCherry-expressing captured cells were washedwith PBS and imaged directly. Staining-positive cells were screenedusing the BioView Ltd. Automated imaging system (Billerica, Mass.).High-resolution pictures were obtained with an upright fluorescencemicroscope (Eclipse 90i, Nikon, Melville, N.Y.).

CTC-iChips were designed and fabricated as previously described (7).Before processing, whole blood samples were exposed to biotinylatedantibodies anti-CD45 (R&D Systems) and anti-CD66b (AbD Serotec,biotinylated in house) and then incubated with Dynabeads® MyOne™Streptavidin T1 (Invitrogen) to achieve magnetic labeling and depletionof white blood cells (7). The CTCs-enriched product was stained insolution with Alexa488-conjugated antibodies anti-EpCAM (Cell SignalingTechnology), anti-EGFR (Cell Signaling Technology), anti-Met (CellSignaling Technology), anti-Cadherin 11 (R&D Systems) and anti-HER2(Biolegend) to identify CTCs, as well as TexasRed-conjugated antibodiesanti-CD45 (BD Biosciences), anti-CD14 (BD Biosciences) and anti-CD16 (BDBiosciences) to identify contaminating white blood cells.

Assessment of metastasis-free survival and overall survival.Kaplan-Meier survival curves based on clinical data from patients atMassachusetts General Hospital were generated with XLStat™ software(Addinsoft). For “Plakoglogin high” vs “Plakoglobin low” distantmetastasis-free survival in breast cancer patients, we used the KM plotresource (available on the World Wide Web at www.kmplot.com) (17).

Single cell micromanipulation. The CTC-enriched product was collected ina 35 mm petri dish and viewed using a Nikon Eclipse Ti™ invertedfluorescent microscope. Single CTCs and CTC-clusters were identifiedbased on intact cellular morphology, Alexa488-positive staining and lackof TexasRed staining. Target cells were individually micromanipulatedwith a 10 μm transfer tip on an Eppendorf TransferMan® NK 2micromanipulator and ejected into PCR tubes containing RNA protectivelysis buffer (10×PCR Buffer II, 25 mM MgCl2, 10% NP40, 0.1 M DTT,SUPERase-In, Rnase Inhibitor, 0.5 uM UP1 Primer, 10 mM dNTP andNuclease-free water) and immediately flash frozen in liquid nitrogen.

Single Cell Amplification and Sequencing. RNA samples extracted fromCTCs were thawed on ice and incubated at 70° C. for 90 seconds. Togenerate cDNA, samples were treated with reverse transcription mastermix (0.05 uL RNase inhibitor, 0.07 uL T4 gene 32 protein, and 0.33 uLSuperScript III Reverse Transcriptase per 1× volume) and incubated onthermocycler at 50° C. for 30 minutes and 70° C. for 15 minutes. Toremove free primers, 1.0 uL of EXOSAP mix was added to each sample,which was incubated at 37° C. for 30 minutes and inactivated at 80° C.for 25 minutes. Next, a 3′-poly-A tail was added to the cDNA in eachsample by incubating in master mix (0.6 uL 10×PCR Buffer II, 0.36 uL 25mM MgCl₂, 0.18 uL 100 mM dATP, 0.3 uL Terminal Transferase, 0.3 uL RNaseH, and 4.26 uL H₂O per 1× volume) at 37° C. for 15 minutes andinactivated at 70° C. for 10 minutes. A second strand cDNA wassynthesized by dividing each sample into 4 and incubating in master mix(2.2 uL 10× High Fidelity PCR Buffer, 1.76 uL 2.5 mM each dNTP, 0.066 uLUP2 Primer at 100 uM, 0.88 uL 50 mM MgSO₄, 0.44 uL Platinum Taq DNAPolymerase, and 13.654 uL H₂O per 1× volume) at 95° C. for 3 minutes,50° C. for 2 minutes, and 72° C. for 10 minutes. PCR amplification (95°C. for 3 minutes, 20 cycles of 95° C. for 30 seconds, 67° C. for 1minute, and 72° C. for 6 minutes 6 seconds) was performed with mastermix (4.1 uL 10× High Fidelity PCR Buffer, 1.64 uL 50 mM MgSO₄, 4.1 uL2.5 mM each dNTP, 0.82 uL AUP1 Primer at 100 uM, 0.82 uL AUP2 Primer at100 uM, 0.82 uL Platinum Taq DNA Polymerase, and 6.7 uL H₂O per 1×volume). The 4 reactions of each sample were pooled and purified usingthe QIAGEN PCR Purification Kit™ (Cat. No 28106) and eluted in 50 uL EBbuffer. Samples were selected by testing for genes Gapdh, ActB, Ptprc(CD45), Krt8, Krt18, Krt19, and Pdxl using qPCR. Each sample was againdivided in 4 and a second round of PCR amplification (9 cycles of 98° C.for 3 minutes, 67° C. for 1 minute, and 72° C. for 6 minutes 6 seconds)was performed with master mix (9 uL 10× High Fidelity PCR Buffer, 3.6 uL50 mM MgSO₄, 13.5 uL 2.5 mM each dNTP, 0.9 uL AUP1 Primer at 100 uM, 0.9uL AUP2 Primer at 100 uM, 1.8 uL Platinum Taq DNA Polymerase, and 59.1uL H₂O per 1× volume). Samples were pooled and purified using AgencourtAMPure XP™ beads and eluted in 40 uL 1× low TE buffer.

Sequencing Library Construction. To shear the DNA using the Covaris S2™System, 1× low TE buffer and 1.2 uL shear buffer were added to eachsample. Conditions of the shearing program include: 6 cycles, 5° C. bathtemperature, 15° C. bath temperature limit, 10% duty cycle, intensity of5, 100 cycles/burst, and 60 seconds. Then, samples were end-polished atroom temperature for 30 minutes with a master mix (40 uL 5× ReactionBuffer, 8 uL 10 mM dNTP, 8 uL End Polish Enzymel, 10 uL End PolishEnzyme2, and 14 uL H₂O per 1× volume). DNA fragments larger than 500 bpwere removed with 0.5× volumes of Agencourt AMPure XP™ beads.Supernatant was transferred to separate tubes. To size-select 200-500 bpDNA products, 0.3× volumes of beads were added and samples were washed2× with 70% EtOH. The products were eluted in 36 uL low TE buffer. AdA-tail was added to each size-selected DNA by treating with master mix(10 uL 5× Reaction Buffer, luL 10 mM dATP, and 5 uL A-Tailing Enzyme Iper 1× volume) and incubated at 68° C. for 30 minutes and cooled to roomtemperature. To label and distinguish each DNA sample for sequencing,barcode adaptors (5500 SOLiD 4464405) were ligated to DNA using the 5500SOLiD Fragment Library Enzyme Module (4464413). Following barcoding,samples were purified twice using the Agencourt AMPure XP™ beads andeluted in 22 uL low TE buffer. Following a round of PCR Amplification(95° C. for 5 minutes, 12 cycles of 95° C. for 15 seconds, 62° C. for 15seconds, and 70° C. for 1 minute, and 70° C. for 5 minutes), thelibraries were purified with AMPure XP™ beads. Finally, to quantify theamount of ligated DNA, SOLiD™ Library TaqMan™ Quantitation Kit was usedto perform qPCR. Completed barcoded libraries were then subjected toemulsion PCR with template beads preparation and sequenced on the ABI5500XL™.

Sequencing data analysis. Determination of reads-per-million (rpm):color space reads were aligned using tophat and bowtie 1 with theno-novel-juncs argument set with human genome version hg19 andtranscriptome defined by the hg19 knownGene table on the World Wide Webat genome.ucsc.edu. Reads that did not align or aligned to multiplelocations in the genome were discarded. The hg19 table knownToLocusLinkfrom genome.ucsc.edu was used to map, if possible, each aligned read tothe gene whose exons the read had aligned to. The reads count for eachgene was the number of reads that were so mapped to that gene. Thiscount was divided by the total number of reads that were mapped to anygene and multiplied by one million to form the reads-per-million (rpm)count. rpm rather than rpkm was used because of a 3′ bias in thealignments.

Clustering: the minimum of 1 and the smallest positive value of the rpmmatrix was added to the rpm matrix to eliminate zeros. The result wasthen log transformed. The result was then median polished. The rows(corresponding to genes) with the top 2000 standard deviations wereretained and the rest of the rows discarded. The result was clusteredusing agglomerative hierarchical clustering with average linkage withdistance metric equal to 1 minus the Pearson correlation coefficient.

Supervised differential gene expression: for each pair of good qualitysingle-cell sample and cluster sample from the same patient, an FDRq-value and a normalized fold-change were calculated using the DEGexpfunction of version 1.10.0 of the Bioconductor DEGseg™ package (18) withmethod set to ‘MARS’ and q-values calculated using Benjamini-Hochberg.For each pair and direction (e.g., up in clusters vs. single-cells) agene was considered a hit if its q-value was less than 0.01 and itsfold-change was greater than 2. Then, for each direction, the genes thatwere hits for 70% or more of the pairs were considered.

Mouse experiments. All mouse experiments were carried out in complianceto the institutional guidelines. For tail vein experiments, NOD SCIDGamma (NSG) mice (Jackson Labs) were injected with 2×10⁵ LM2 cells andmonitored with IVIS® Lumina II™ (Caliper LifeSciences). For CTC-clustersmetastatic potential assessment and intravital imaging, 2×10⁶ LM2-GFPand 2×10⁶ LM2-mCherry cells were mixed 1:1, suspended in 100 μl of 50%Basement Membrane Matix Phenol Red-free (BD Biosciences) in PBS andinjected orthotopically in NSG mice. Intravital imaging, as well asblood draw for CTCs enumeration, was performed 5 weeks after tumoronset. For Plakoglobin knockdown experiments, 1×10⁶ LM2-CTRL orLM2-Plakoglobin shRNA cells were suspended in 100 μl of 50% BasementMembrane Matix Phenol Red-free in PBS and injected orthotopically in NSGmice. Blood draw for CTCs enumeration and lung metastasis analysis wereperformed 4 weeks after tumor onset.

Intravital Imaging and In Vivo Flow Cytometry. For intravital imaging,mice were anesthetized with 1.3% isoflurane and the mammary tumor wassurgically exposed to provide optic access. The mice were put on amotorized stage and 2% methocellulose (Methocel 2%, OmniVision) alongwith a #1 coverglass were applied to the tumor site. The draining bloodvessels directly next to and within the primary tumor were scanned witha video-rate confocal microscope (19). Appropriate locations for imagingwere determined and video-rate movies were recorded. GFP and mCherryproteins were excited with 491 nm and 561 nm lasers, respectively, andthe fluorescence was detected by photomultiplier tubes (R3896, HamamatsuPhotonics) equipped with confocal pinholes and 528±18 nm and 593±20 nmbandpass filters, respectively (19). A confocal reflectance channel wasalso recorded using a 635 nm laser and a third photomultiplier tube. Thereflectance channel allowed the delineation of flowing blood vesselswithout introduction of an exogenous contrast agent.

For in vivo flow cytometry, DiD-labeled LM2 cells were adoptivelytransferred intravenously and detected in the peripheral circulation(20). DiD was excited by a 635 nm laser and detected with a 695±27.5 nmbandpass filter using a photomultiplier tube. Circulation kinetics forLM2-SCs and LM2-CLs were quantified using MATLAB™ (Mathworks).

Immunohistochemistry. Formalin-fixed and paraffin embedded mouse primaryLM2 tumors and lung metastases, as well as human primary tumors andmatched metastatic lesions were sectioned and stained overnight at 4° C.with antibodies anti-cleaved caspase 3 (Cell Signaling Technology),anti-GFP (Cell signaling Technology), anti-mCherry (Abcam),anti-Plakoglobin (Sigma Aldrich) and anti-CD31 (Abcam). GFP/mCherry andPlakoglobin/CD31 double-stainings were performed with EnVision™ G/2Doublestain System (Dako). All specimens were counterstained withHematoxilyin. Images of the whole tissue were taken with ScanScope™(Aperio).

Cell culture and reagents. HMEC, MCF10A, BT474, MCF7, T47D, BT549, BT20and ZR-75-1 cells were purchased from the American Type CultureCollection (ATCC) and propagated according to the manufacturer'sinstructions. MDA-MB-231 LM2 cells were propagated in DMEM (LifeTechnologies) supplemented with 10% fetal bovine serum (LifeTechnologies). To generate LM2 single cells (LM2-SCs) or LM2 clusters(LM2-CLs) for tail vein injections, LM2 cells growing in monolayer at80% confluence were incubated with trypsin (Life Technologies) for oneminute to generate floating LM2-CLs. LM2-CLs were then distributedequally in two separate dishes. In one of the two dishes, LM2-CLs weremechanically dissociated by pipetting to generate a single cellsuspension of LM2-SC2.

Cell-to-cell adhesion assay was performed with the Vybrant® Cell-to-CellAdhesion Assay Kit (Invitrogen) according to the manufacturer'sinstructions.

The plasmid expressing GFP-Luciferase was obtained from C. Ponzetto(University of Torino, Italy). The plasmid expressing mCherry waspurchased from Addgene. Plakoglobin TRC shRNAs were purchased fromThermo Scientific. Lentiviral packaging vectors (Addgene) were used totransfect 293T cells (ATCC) and produce lentiviral particles. Infectionsof target cells lines was performed overnight at a MOI=10 in growthmedium containing 8 μg/ml polybrene (Thermo Scientific).

TABLE 1 Progression-free survival, age, tumor subtype, and mutationstatus of single CTC-enriched and CTC-clusters-enriched breast cancerpatients. Patient Progression-free Mutation ID survival Age Subtypestatus SINGLE CTCs-ENRICHED PATIENTS BRX02 91 42 TN8C PI3K mutationBRX09 139 40 HR+ BRCA mutation BRX10 85 52 HR+ No BRx12 102 57 HR+ NoBRX13 768 60 HR+ HRAS and PI3K Mutation BRX14 51 51 TNBC Negative BRX1728 66 HR+ PI3K Mutation BRX18 448 53 HER2+ PI3K mutation BRX21 71 78 HR+Snapshot not done BRX22 66 53 HER2+ PI3K mutation BRX23 42 77 TNBC PI3Kmutation BRX27 62 63 HR+ No BRX34 258 66 TNBC P53 BRX35 71 54 HR+Snapshot not done BRX36 30 43 TNBC No BRX40 336 76 HR+ PI3K MutationBRX42 247 54 TNBC PI3K Mutation BRX43 112 70 HR+ No BRX44 125 51 HER2+No BRx64 172 48 HR+ No BRx72 67 44 HER2+ PI3K mutation BRX24 111 65 TNBCNo BRX26 263 74 HR+ No BRX47 185 35 HER2+ No BRX55 85 80 HR+ PI3Kmutation CTC-CLUSTERS-ENRICHED PATIENTS BR16 104 73 HR+ PI3K and KRASmutation BR18 42 32 HR+ TP53 BRX07 41 75 HR+ No BRX38 168 54 HR+ PI3Kmutation BRx50 22 42 HR+ BRCA mutation BRX53 122 53 HR+ PI3K mutationBRx61 34 54 HR+ No

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Example 2

Desmosome and adherence junction genes are upregulated in CTC-C cells(FIGS. 10A-10D), as compared to, e.g. CTC cells.

Example 3: Circulating Tumor Cell Clusters Are Oligoclonal Precursors ofBreast Cancer Metastasis SUMMARY

Circulating tumor cell clusters (CTC clusters) are present in the bloodof patients with cancer but their contribution to metastasis is not welldefined. Using mouse models with tagged mammary tumors, it isdemonstrated herein that CTC clusters arise from oligo-clonal tumor cellgroupings and not from intravas-cular aggregation events. Although rarein the circulation compared with single CTCs, CTC clusters have 23- to50-fold increased metastatic potential. In patients with breast cancer,single-cell resolution RNA sequencing of CTC clusters and single CTCs,matched within individual blood samples, identifies the cell junctioncomponent plakoglobin as highly differentially expressed. In mousemodels, knockdown of plakoglobin abrogates CTC cluster formation andsuppresses lung metastases. In breast cancer patients, both abundance ofCTC clusters and high tumor plakoglobin levels denote adverse outcomes.Thus, CTC clusters are derived from multicellular groupings of primarytumor cells held together through plakoglobin-dependent intercellularadhesion, and though rare, they greatly contribute to the metastaticspread of cancer.

INTRODUCTION

The metastatic spread of breast cancer, typically to bone, lung, liver,and brain, accounts for the vast majority of cancer-related deaths(Nguyen et al., 2009). Our understanding of epithelial cancer metastasisis derived primarily from mouse models and it is thought to involve aseries of sequential steps: epithelial-to-mesenchymal transition (EMT)of individual cells within the primary tumor leading to theirintravasation into the bloodstream, survival of such circulating tumorcells (CTCs) within the bloodstream, and finally their extravasation atdistant sites, where mesenchymal-to-epithelial transition (MET)culminates in their proliferation as epithelial metastatic deposits(Hanahan and Weinberg, 2011). While EMT has indeed been demonstrated inhuman breast cancer cells in the circulation (Yu et al., 2013), therequirement for EMT to initiate metastasis has been debated (Ledford,2011; Tarn et al., 2005). Alternative models proposed includetumor-derived microemboli that may break off from primary tumors,lodging into distal capillaries where they initiate metastatic growth(Fidler, 1973; Liotta et al., 1976; Molnar et al., 2001). Using diversetechnological platforms, we and others have indeed detected clusters ofCTCs, ranging from 2-50 cancer cells, within the circulation of patientswith metastatic epithelial cancers (Cho et al., 2012; Fidler, 1973;Molnar et al., 2001; Stott et al., 2010; Yu et al., 2013).

Studies of cancer metastasis have emphasized the concept of “seed versussoil” as a key determinant of metastatic propensity (Fidler, 2003). Thismodel matches the importance of mutated genetic drivers within tumorcells conferring proliferative and invasive properties, with that of themicroenvironment of the distant organ or “niche,” which may facilitatemetastatic growth. However, the physical characteristics of single CTCsand CTC clusters may also contribute to metastatic propensity,especially as they impact the ability of epithelial tumor cells tosurvive the loss of cell adherence and shear forces in the blood stream,i.e., different survival signals among the cancer cell “seeds” may beimportant. For instance, in a mouse endogenous pancreatic cancer model,noncanonical Wnt signaling is elevated within CTCs, where it appears tosuppress anoikis (Yu et al., 2012), while in a subcutaneous tumorxenograft model, the admixture of tumor and stromal cells withinmicroemboli may contribute stromal-derived survival signals (Duda etal., 2010).

CTCs have been detected in the majority of epithelial cancers, wherethey represent cancer cells captured as they transit through thebloodstream (Alix-Panabiéres and Pantel, 2013; Yu et al., 2011). Assuch, they hold the key to understanding critical pathways that mediatethe bloodborne dissemination of cancer, which may not be readily evidentthrough analyses of bulk primary or metastatic tumor populations.Factors leading to the generation of CTCs from a primary tumor areunknown, including the fraction derived from cancer cells that haveactively intravasated into the bloodstream, versus those that arepassively shed as a result of compromised tumor vasculature. Althoughexceedingly rare compared with normal blood cells, the number of CTCs inthe bloodstream far exceeds the number of metastatic lesions inpatients, indicating that the vast majority CTCs die in the bloodstream,with only a minor fraction representing viable metastatic precursors.Epithelial cells that have lost adhesion-dependent survival signalsrapidly undergo anoikis, a fate likely to meet most CTCs in thebloodstream. It is in this context that either mesenchymaltransformation, stromal-derived factors, or persistent interepithelialcell junctions may provide survival signals that attenuate thisapoptotic outcome (Duda et al., 2010; Mani et al., 2008; Robson et al.,2006; Yu et al., 2012). Dissecting the contributions of these variousmechanisms to human cancer requires the ability to isolate individualCTCs from the bloodstream and subject these to detailed molecularanalyses.

Multiple technologies have been developed for CTC capture, takingadvantage of tumor-specific epitopes absent in normal blood cells,variations in their physical properties such as size, density, andelectromechanical characteristics, or by applying high throughputimaging to unpurified blood cell preparations (for review, see Yu etal., 2011). The fact that CTCs are extremely rare, even in patients withadvanced metastatic cancers (estimated at one CTC/billion normal bloodcells), and that they may be poised on the verge of apoptosis, has madetheir analysis contingent upon technological constraints. We haveintroduced a series of microfluidic devices that have the advantage oflow-shear, yet high throughput, interrogation of unprocessed wholeblood, providing highly enriched and unfixed CTCs that are suitable fordetailed molecular analysis (Nagrath et al., 2007; Ozkumur et al., 2013;Stott et al., 2010). Among these, the herringbone (HBCTC-Chip) makes useof grooves within the ceiling of the microfluidic chamber to generateturbulent micro-fluidic flow, directing cells against antibody-coatedwalls of the device, where CTCs are captured (Stott et al., 2010). Thisdevice, whose highly efficient design enabled the initial detection oflarge CTC clusters, requires on-chip cell lysis for nucleic acidextraction and hence provides an enriched but heterogeneous CTCpopulation for analysis (Yu et al., 2012, 2013).

In contrast, our recently described negCTC-iChip achieves highlyefficient depletion of erythrocytes and leukocytes from blood specimens,yielding untagged CTCs and small CTC clusters in solution, where theycan be micromanipulated for single-cell RNA sequencing (Ozkumur et al.,2013). The experiments described herein utilize both of these devices,along with in vivo flow cytometry and next generation RNA sequencing, tointerrogate CTCs from both patients with metastatic breast cancer andmouse tumor models. As described herein, using mouse models, CTCclusters are derived from oligo-clonal clumps of primary tumor cells andconstitute a rare but very highly metastasis-competent subset of CTCs,compared with single circulating breast cancer cells. RNA sequencing ofhuman breast CTC clusters identifies plakoglobin as a key mediator oftumor cell clustering, which is expressed in a heterogeneous patternwithin the primary tumor. Knockdown of plakoglobin expression in themouse model suppresses CTC cluster formation and reduces metastaticspread.

Results

Endogenous CTC Clusters Have Increased Metastatic Potential Compared toSingle CTCs

To define the origin and functional properties of CTC clusters, comparedwith single CTCs, mouse models were utilized, where tumor cellcomposition, transit of CTCs through the blood-stream, and metastaticdeposits can be monitored and quantified. First, a model was establishedto test the generation of endogenous CTCs and metastases from a primaryorthotopic tumor xenograft. These experiments were designed both to testthe metastatic propensity of CTC clusters versus single CTCs, as well asto determine whether CTC clusters originate from an oligoclonal groupingof primary tumor cells or from the clonal progeny of an individual tumorcell. MDA-MB-231-LM2 (LM2) cells, a lung-metastatic variant ofMDA-MB-231 human breast cancer cells (Minn et al., 2005), wereengineered to express either green fluorescent protein (LM2-GFP) ormCherry (LM2-mCherry), and a 1:1 mixture of these differentially taggedcells was injected into the mammary fat pad of immunodeficient (NSG)mice. As expected, overt primary breast tumors were observed after 5weeks and these retained an equal distribution of LM2-GFP andLM2-mCherry tagged cells, as confirmed by IHC staining (data not shown).The blood of tumor-bearing animals was sampled for presence of single orclustered CTCs using a terminal bleed and the lungs were simultaneouslyharvested for analysis of metastatic deposits. In addition toenumeration of CTCs, it was reasoned that clonally-derived CTC clusterswould uniformly express either GFP or mCherry, whereas aggregations ofcells from the primary tumor would be heterogeneous for the two markers(FIG. 11A). A mean of 2,486 CTC events per mouse (n=5 mice) wasobserved, of which a mean of 65 (2.6%) were CTC clusters and 2,421(97.4%) were single CTCs (FIGS. 11B and 17A). Virtually all (91%) CTCclusters were dual positives for GFP and mCherry. A mean of 5.6 (9%) CTCclusters per mouse (with fewer than three cells per cluster) werecomprised of cells expressing only one of the two markers, consistentwith expected probabilities given a 1:1 mixture of GFP/mCherryexpressing cells in the primary tumor (FIGS. 11B and 17B). Thus, CTCclusters do not result from the proliferation of a single tumor cell inthe vasculature, insteadthey appear to represent the aggregation ofneighboring cells, most likely within the primary tumor mass (seebelow).

Metastatic deposits in the lungs were analyzed for both number andcomposition using anti-GFP and anti-mCherry antibodies, simultaneouslywith the CTC analyses (FIGS. 11B and 17A). Given the distribution of GFPand mCherry staining in CTC clusters, it was reasoned that metastatictumors derived from a single CTC would be positive for a single marker,while those derived from CTC clusters would stain for both GFP andmCherry (FIG. 11A). A mean of 323 lung foci were identified per mouse(n=5 mice), of which 171 (53%) were multicolor, and therefore derivedfrom CTC clusters, versus 152 (47%) unicolor derivatives of single CTCs(FIGS. 11B and 17A). Normalizing the number and distribution of lungmetastases with that of single CTCs and CTC clusters, it was calculatedthat a CTC cluster is 50 times more likely to give rise to a metastaticdeposit than a single CTC (FIG. 11C). Thus, while CTC clusters are muchmore rare than single CTCs in this orthotopic mouse model of breastcancer, they contribute equally to the metastatic burden in the lung.

To further validate (1) that oligoclonal CTC clusters arise from thefragmenting of primary tumor cell clumps into the vasculature and notfrom intravascular aggregation of single CTCs, and (2) that oligoclonallung metastases arise from CTC clusters and not from the reseeding of ametastatic site by multiple single CTCs, a second series of orthotopicmouse xenograft experiments was conducted, injecting LM2-GFP cells intothe right mammary fat pad and LM2-mCherry cells in the left fat pad ofimmunodeficient mice (FIG. 11D). Five weeks after injection, miceharbored two independent and differentially tagged tumors, and the bloodwas harvested for analysis of CTCs and the lungs for enumeration ofmetastatic deposits. As expected, single CTCs in the circulationdemonstrated equal contributions from the GFP and m-Cherry-taggedprimary tumors. However, unlike the previous multitagged single tumormodel, in mice with two independent individually-tagged tumors, the vastmajority of CTC clusters (96%) were of a single color, with equalcontributions from GFP- or mCherry-positive primary tumors (FIGS. 11Eand 17A). Thus, the vast majority of CTC clusters are derived fromindividual primary tumors, excluding intravascular aggregation of singleCTCs as a significant source of CTC clusters.

A very small fraction of CTC clusters observed in the dual tumor-bearingmice were multicolor (4% of CTC clusters, corresponding to 0.12% oftotal CTC events) (FIGS. 11E and 17A). While extraordinarily rare, thepresence of such CTC clusters derived from two independent tumors mayoriginate either from the uncommon intravascular aggregation of singleCTCs or from a mixing of cancer cells within the two primary tumors, dueto the previously reported “tumor reseeding” phenomenon (Kim et al.,2009) (data not shown). Consistent with the latter hypothesis, it wasfound that 3%-5% of cells within the GFP-tagged primary tumor werepositive for mCherry and 3%-5% of cells within the mCherry-labeled tumorwere positive for GFP (data not shown). In addition to rare multicolorCTC clusters, a small fraction (8%) of multicolor tumors in the lung wasobserved (FIGS. 11E and 17A). These metastatic lesions could resulteither from the rare multicolor CTC clusters or from the reseeding ofmetastatic lesions by multiple single CTCs.

The findings derived from the two LM2 mouse xenograft experiments wereconfirmed with a second, mouse-derived breast cancer cell line, 4T1(FIGS. 17C and 17D). Consistent with the LM2 results, a 1:1 mixture of4T1-GFP and 4T1-mCherry cells within an orthotopic mammary tumorgenerated CTC clusters that were overwhelmingly multicolored (90%),whereas two separate primary 4T1 tumors labeled either with GFP ormCherry produced CTC clusters that were of a single color (87%). Theseobservations further support that CTC clusters arise as oligoclonalfragments derived from a single tumor (FIG. 17C). Normalizing the numberand color distribution of 4T1-derived lung metastases relative to theprevalence of single CTCs and CTC clusters, a 23-fold increase inmetastatic competence for CTC clusters versus single CTCs was calculated(FIG. 17D), an estimate that is comparable to the 50-fold increasederived from LM2 cell experiments. Taken together, these two mouse tumormodels indicate that CTC clusters constitute only 2%-5% of all CTCevents detected in the circulation, but their dramatically elevatedmetastatic potential (23-50 times that of single CTCs) contributes toapproximately half of all metastatic lesions in orthotopic breast cancermodels.

Clustered Cancer Cells Are More Resistant than Single Cells to Apoptosisfollowing Dissemination to the Lung

An in vitro assay was generated that allowed us to obtain a suspensionof either single cells or clustered cells (2-30 cells) from cultures ofGFP-Luciferase-tagged LM2 cells (see Extended Experimental Procedures).200,000 LM2 cells prepared either as single cells (LM2-SC) or asclusters (LM2-CL) were injected into the tail vein of immunodeficientmice and the mice then subjected to serial luciferase-based imaging(FIG. 12A). Both LM2-SC and LM2-CL cells reached the lungs with equalefficiency (day 0), as shown by both bioluminescence and GFPimmunohistochemical (IHC) staining (FIG. 12A). However, over thefollowing days, the LM2-SC lung signal progressively diminished as thecells underwent massive apoptosis, demonstrated by staining for cleavedcaspase 3 (FIG. 12B). In contrast, the LM2-CL lung signal persistedfollowing intravascular inoculation, with cells showing resistance toapoptosis and tumors expanding more rapidly (FIGS. 12A, 12B, and 2A).Lung tumors eventually grew in mice subjected to tail vein injectionwith either of the two LM2 derivatives, but injection of clustered cellsresulted in reduced overall survival, with 12.7 weeks for LM2-CL versus15.7 weeks for LM2-SC (p<0.016) (FIG. 6B). The differential rate ofapoptosis and metastatic growth in the lung was confirmed for singleversus clustered cancer cells using tail vein injection of twoadditional breast cancer cell lines, BT474 and 4T1 (FIGS. 18A-18C).

Calculation of CTC Clusters and Single CTC Circulatory Clearance RateUsing In Vivo Flow Cytometry

Clusters of tumor cells may exhibit considerable flexibility as theynavigate through narrow channels, and capillary beds themselves may haveuneven vessel diameters or bypass tracts that allow transit of largemulticellular structures. However overall, CTC clusters are more likelythan single CTCs to be trapped in small capillaries of the lung anddistal organs. Thus, the low steady-state level of CTC clusters in thecirculation may reflect a considerably higher generation rate if theirclearance rate is very high. To test if CTC clusters indeed have afaster clearance rate from the bloodstream than single CTCs, in vivoflow cytometry (IVFC) was used to monitor LM2-SC and LM2-CL cellslabeled with the lipophilic carbocyanine membrane dye DiD, followingtail vein injection in immunodeficient mice (FIG. 13). DiD was selectedto achieve optimal detection of CTCs with the IVFC settings. CirculatingDiD-labeled cells were detected in real time within the ear bloodvessels for a total of 55 min in each mouse. Injected LM2-CL cells werecleared at least three times more rapidly than LM2-SC (half-life: 6-10min for LM2-CL versus 25-30 min for LM2-SC) (FIG. 3). Together, theseobservations define a circulating time for CTCs in the bloodstream: theshorter circulation half-life of CTC clusters is consistent with theirmore rapid entrapment within capillaries of distal organs, where theymay initiate metastatic growth (Liotta et al., 1976).

The Presence of CTC Clusters in Patients with Breast and Prostate CancerCorrelates with Poor Prognosis

Having characterized the origin and metastatic potential of CTC clustersin mouse models, it was undertaken to study their properties in patientswith cancer. To first test the clinical significance of CTC clusters inthe blood of patients with progressing metastatic breast cancer, theirpresence was measured in blood specimens from a total of 79 patients,drawn at multiple time points over a period of 19 months. Patients wererecruited to an IRB-approved study at the Massachusetts General HospitalCancer Center, including women with estrogen receptor-positive (n=49),HER2-positive (n=13), triple negative (n=17) subtypes of breast cancer(total: 265 data points). For these experiments, the HBCTC-Chip wasutilized, which is highly efficient in capturing both large and smallCTC clusters (Stott et al., 2010). The microfluidic chamber was coatedwith a combination of antibodies, targeting the epithelial cell adhesionmolecule (EpCAM), as well as the line-age markers epithelial growthfactor receptor (EGFR) and human epithelial growth factor receptor 2(HER2/ErbB2), which together efficiently capture both epithelial andmesenchymal breast CTCs (Yu et al., 2013). After processing 3 ml ofwhole blood from patients with breast cancer, the CTCs captured on thechip were stained with antibodies against wide spectrum cytokeratin (CK)to identify CTCs and against the leukocyte marker CD45 to assess whiteblood cell (WBC) contamination (data not shown). CTCs were identified in54 out of 79 patients (68%). Among patients with CTCs, 3 (5.6%) had CTCclusters evident across more than three time points, while 16 (29.6%)had CTC clusters during one to three time points and 35 (64.8%) had nodetectable clusters (FIG. 14A). The presence of CTC clusters withprogression-free survival (PFS) was calculated for all patients wheresuch data were available (n=30). Of note, PFS was calculated as timefrom initiation of therapy to discontinuation by the treating clinician(blinded to the CTC results), and PFS data analysis was performed onlywhen clinical measurements bracketed the CTCs isolation time frame.Patients with CTC clusters across more than three time points had a meanprogression-free survival time of 32.6 days, compared with 134.8 daysfor patients where CTC clusters were found during one to three timepoints and 160.5 days for patients with single CTCs only (p=0.0002)(FIG. 14B). Thus, even among patients with advanced metastatic breastcancer, the continuous presence of CTC clusters is associated with anadverse clinical outcome.

Given the relatively short time to progression in patients with advancedbreast cancer, it was sought to test the correlation between CTCclusters and adverse prognosis in patients with a longer clinicalcourse. The number of CTCs was measured in a total of 64 patients withprostate cancer using blood specimens drawn at multiple time points overa period of 53 months (total: 202 data points). CTCs in prostate cancerpatients were visualized by staining with a cocktail of antibodiesagainst prostate-specific antigen (PSA) and prostate-specific membraneantigen (PSMA); anti-CD45 staining was used to exclude white blood cells(Miyamoto et al., 2012). CTCs were detected in 48/64 patients (75%). CTCclusters were present in 6/48 samples (12.5%) (FIG. 14C). In thiscohort, the presence of CTC clusters during at least one time pointstrongly correlated with a dramatically shorter overall survival time(mean survival time was 115.8 days for patients with CTC clusters versus930.1 days for patients with single CTCs; p=0.00001) (FIG. 14D). Theseresults indicate the relevance of CTC clusters in the progression ofhuman cancer.

Single-Cell Resolution RNA Sequencing of Matched CTC Clusters and SingleCTCs Purified from Patients with Breast Cancer

The ability to capture both single CTCs and CTC clusters from the sameblood specimen made it possible to undertake single-cell resolution RNAsequencing, searching for differences in expression profiles matched toindividual patients. For these experiments, the negCTC-iChip wasapplied, which enables isolation and single-cell manipulation ofuntagged CTCs, together with an optimized protocol for next generationRNA sequencing from minute amounts of template (Ozkumur et al., 2013;Tang et al., 2010). Blood specimens from ten patients with metastaticbreast cancer were subjected to microfluidic depletion of RBCs and CD45-and CD66b-positive WBCs, leaving untagged single CTCs and small CTCclusters in the final product (Ozkumur et al., 2013). Unfixed tumorcells were stained for cell surface expression of EpCAM, HER2, and themesenchymal marker CDH11 (Alexa488-conjugated), and counterstained withantibodies against CD45, CD14, and CD16 to identify contaminatingleukocytes (TexasRed-conjugated) (FIG. 4A). Individual CTC clusters(median of three cells per cluster) were isolated using amicromanipulator and compared with numerically matched pools of singleCTCs from the same specimen, followed by next generation RNA sequencing(SOLiD 5500XL) (FIG. 4A). Normalized expression profiles were derivedfor a total of 29 samples (15 pools of single CTCs and 14 CTC clusters)isolated from ten breast cancer patients.

Unsupervised hierarchical clustering of RNA sequencing data showed noobvious distinctions at the global gene expression level between singleCTCs and CTC clusters, with both of these clustering closely by patientof origin (FIG. 4B). Consistent with the microscopic appearance of CTCclusters as primarily tumor cell-derived, RNA signatures of other celltypes were not identified, including T cells, B cells, dendritic cells,natural killer cells, hematopoietic stem cells, macrophages/monocytes,granulocytes, endothelial cells, or fibroblasts (FIG. 19). Markers forplatelets were present in both single CTCs and CTC clusters, consistentwith their known adherence to cancer cells in the circulation. For eachpatient, gene expression data of CTC clusters versus single-CTCs wascompared, generating a list of 31 CTC-cluster-associated genes sharedacross different patients (q<0.01, log 2FC>1, in more than 70% of allintrapatient comparisons) (FIGS. 4C, 4D and 9). To identify potentialdrivers of metastasis among CTC-cluster-enriched genes, correlationbetween their overexpression in primary tumor specimens and clinicaloutcomes was tested in a cohort of 1,956 patients with ER-positive,HER2-positive, and triple-negative breast cancers. Among the candidateCTC cluster genes, plakoglobin was unique in its high level ofoverexpression in CTC clusters compared with single CTCs (219-fold) andthe fact that its expression in primary tumors associated with asignificantly reduced distant metastasis-free survival (p=0.008) (FIGS.4D, 5E, and 20). Plakoglobin was therefore selected as aCTC-cluster-enriched transcript for more detailed analysis.

Plakoglobin (JUP) is a member of the Armadillo family of proteins and animportant component of desmosomes and adherence junctions (Aktary andPasdar, 2012), which has been reported to have both positive andnegative roles in diverse malignancies (Hakimelahi et al., 2000; Kolligset al., 2000; Shiina et al., 2005). Along with upregulation ofplakoglobin RNA, multiple components of both desmosomes and adherencejunctions were significantly enriched in CTC clusters (FIGS. 10A-10D).Consistent with the RNA sequencing results, plakoglobin proteinexpression was confirmed in multiple CTC clusters, but not in matchedsingle CTCs from a breast cancer patient (data not shown). While CTCclusters express epithelial cell junction components, includingplakoglobin and E-cadherin, some mesenchymal markers may also beupregulated in such clusters, an effect that may be associated withadherence in the bloodstream with TGFj3-rich platelets (Labelle et al.,2011; Yu et al., 2013). Matched primary and metastatic tumors biopsieswere available from this patient: plakoglobin expression was remarkablyheterogeneous in both the primary and metastatic breast tumors, withfoci of high expression interspersed with regions without detectableprotein (FIG. 15). Thus, while plakoglobin is a key component ofintercellular junctions, its variable expression levels within primarytumors raises the possibility that it might demarcate tightly adherentgroups of cells that may constitute precursors to CTC clusters.

Plakoglobin Is Required for CTC Cluster Formation and Contributes toBreast Cancer Metastasis

To define the functional consequences of plakoglobin expression in thecontext of CTC clusters, an in vitro assay (Vybrant), which utilizes afluorogenic dye to measure cell-to-cell adhesion under a variety ofculture conditions was applied (El Khoury et al., 1996). Seven breastcancer cell lines (MDA-MB-231-LM2, BT474, MCF7, T47D, BT549, BT20, andZR-75-1) were compared with two nontransformed human mammary epithelialcells (HMEC and MCF10A), following stable lentiviral-mediatedplakoglobin knockdown. shRNA-mediated plakoglobin suppression triggereddisruption of cell-cell contacts in 6/7 breast cancer lines grown as amonolayer, while it had no detectable effect in either of the twonontransformed breast epithelial cells (p<0.04) (FIGS. 3A, and 8A).Thus, breast cancer cells may be more dependent on plakoglobin-mediatedcell junctions than normal epithelial cells, which may benefit fromadditional or alternative pathways in forming intercellular connections(Alford and Taylor-Papadimitriou, 1996; Cavallaro and Christofori,2004).

To extend these observations in vivo, either plakoglobin shRNAs ornontarget controls were introduced into GFP-Lucif-erase-tagged LM2 andBT474 cells and these prepared as single cells (SC) or clusters (CL) fortail vein injection into immunosuppressed mice. Consistent with theresults described above herein, both LM2 and BT474 cells expressingcontrol shRNAs showed dramatically increased persistence in the lungwhen prepared under CL versus SC conditions. In contrast, despite CLconditions, plakoglobin knockdown in both LM2 and BT474 cellsdissociated clusters into single cells, consistent with the requirementfor plakoglobin for intercellular adhesion in these cells. Followingplakoglobin knockdown, tail vein inoculation of CL and SC preparationsof both LM2 and BT474 were comparable in producing a reduced number oflung metastases (FIG. 16). Thus, plakoglobin knockdown abrogatesintercellular interactions required to generate clustered cancer cells,thereby reducing their potential to produce lung foci after directintravas-cular injection.

Finally, orthotopic xenografts were generated by injectingLM2-GFP-Luciferase cells expressing either control or plakoglobin shRNAsinto the mammary fat pad of immunodeficient mice and measuring tumorgrowth as well as tumor-derived CTCs. Plakoglobin knockdown did notalter the primary tumor growth rate, measured for up to 30 days (FIGS.5B and 8B), nor did it affect the total number of single CTCs derivedfrom the primary tumor (FIG. 5C). Remarkably, the number oftumor-derived CTC clusters was significantly reduced in mice bearing LM2plakoglobin shRNA-expressing tumors compared to control mice (FIG. 5C).In parallel, bioluminescence imaging of mouse lungs demonstrated astriking 80% reduction in lung nodules for mice bearingplakoglobin-suppressed primary tumors (FIG. 5D).

Together, these data indicate a model whereby plakoglobin-expressingregions within a primary tumor produce aggregated tumor cells, i.e., CTCclusters, that are shed into the bloodstream, where they demonstraterapid clearing at distant sites and enhanced metastatic potential (FIG.5F). It is contemplated herein that CTC clusters can be targetedtherapeutically through disruption of cell-cell junctions, e.g.,permitting a reduction of the metastatic spread of breast cancer.

DISCUSSION

By applying microfluidic CTC isolation technologies to both patientswith breast cancer and mouse models, CTC clusters, a striking but poorlyunderstood feature of bloodborne metastasis have been characterized, asdescribed herein. CTC clusters occur in cancers of various origins.While most clusters are relatively small, some comprise dozens of tumorcells, raising the question of how they navigate through normalcapillaries. The in vivo flow cytometry studies indicate that clustersare more rapidly cleared from the circulation than single CTCs.Nonetheless, both the structural deformability of the aggregated cellswithin these clusters and the presence of vascular shunts within thecirculation may allow a subset of these to circulate. The rapidclearance of clusters within distal tissues, together with theirpotentially increased cellular viability may underlie their dramaticallyenhanced metastatic potential. The increased metastatic propensity ofCTC clusters in reconstituted mouse models, together with the adverseprognosis of breast and prostate cancer patients with abundant CTCclusters, support an important role for these cellular aggregates in theblood-borne spread of cancer.

Based on cellular tagging and mixing studies in the mouse, almost allCTC clusters appear to be of oligoclonal origin, rather than beingderived from the progeny of a single migratory cell. The present studiesexclude intravascular aggregation of CTCs as a significant cause for CTCclusters, demonstrating instead that they originate from a single tumor.

Interestingly, the high expression of plakoglobin within foci of cellswithin the primary tumor raises the possibility that these demarcate theorigin of clusters that ultimately enter the circulation. In mousereconstitution models, plakoglobin knockdown in cells that constitutethe primary tumor does not suppress tumorigenesis itself, but itabrogates the generation of CTC clusters in the circulation and greatlyreduces the number of metastatic deposits in the lung.

The identification of specific transcripts that enhance the metastaticpotential of tumor cells, as described herein, permits therapeuticstrategies to suppress the bloodborne spread of cancer, a criticalalthough challenging goal. To date, candidate metastasis genes have beenderived primarily from mouse tumor models. Some, like inducers of EMT,alter the migratory properties of breast cancer epithelial cells andconfer stem-like properties (Mani et al., 2008). In human breast cancerCTCs, we recently documented marked enrichment for mesenchymaltranscripts in CTCs, using quantitative RNA-in situ hybridization (Yu etal., 2013). In addition to generalized migratory properties associatedwith EMT, tissue-specific tropism studies in the mouse have identifiedsubsets of genes involved in breast cancer metastases to lung (e.g.,Epiregulin, CXCL1, SPARC, and MMP2) (Minn et al., 2005), brain (e.g.,COX2, HBEGF, and ST6GALNACS) (Bos et al., 2009), and bone (mainly drivenby Src activation) (Zhang et al., 2009). A recent study interrogatingcandidate genes in breast CTCs derived from a patient with breast cancerhas suggested that coexpres-sion of EpCAM, CD44, CD47, and METidentifies a subset with increased metastatic capacity (Baccelli et al.,2013). These candidate metastasis genes were not upregulated in CTCclusters compared with single CTCs.

The present study identifies mediators of metastasis by comparing twodistinct populations of circulating tumor cells, one with very highmetastatic potential (CTC clusters) compared with the other (singleCTCs). The development of advanced microfluidic CTC isolation technology(Ozkumur et al., 2013) enabled the undertaking of such a detailed studyof human breast cancer cells as they transiently circulate in thebloodstream of patients with metastatic disease. Single-cell resolutionRNA sequencing demonstrated a very high level of concordance inexpression patterns between matched CTC clusters and single CTCs fromindividual breast cancer patients. A number of candidate genes withsignificantly divergent expression were identified (FIG. 9), includingtranscriptional regulators (XBP1), signaling molecules (AGR2 and HER3),and plakoglobin. While we focused this study on the functionalcharacterization of plakoglobin due to the clinical association betweenhigh plakoglobin expression and adverse outcome in patients with breastcancer, additional CTC-cluster-associated genes can be involved in theirgeneration and their metastatic potential. The striking consequences ofplakoglobin knockdown, suppressing both CTC cluster generation andmetastatic tumor formation in mouse models, point to this gene productbeing a major determinant of tumor dissemination. Plakoglobincontributes to both adherens junctions and desmosomes: in adherensjunctions, the C-terminal intracellular domain of E-cadherin interactsin a mutually exclusive manner with either 3-catenin or plakoglobin,which in turn associates with the actin-binding protein a-catenin(Harris and Tepass, 2010). At desmosomes, the intracellular domains ofdesmocolin and desmoglein interact with plakophilin and plakoglobin,which in turn binds the intermediate filament binding proteindesmoplakin (Garrod and Chidgey, 2008). Thus, plakoglobin is a criticalconstituent of both adherens junctions and desmosomes, a role that mayunderlie its unique contribution to cell-to-cell adhesion in tumorcells. While plakoglobin has been implicated as both oncogene and tumorsuppressor in different contexts (Ha-kimelahi et al., 2000; Kolligs etal., 2000; Shiina et al., 2005), it is neither in the model proposedhere, functioning instead as an intercellular tether that confers addedmetastatic potential to tumor cells as they break off into thecirculation. Interestingly, plakoglobin knockdown has far less impact onintercellular connections of nontransformed breast epithelial cells,which may benefit from additional adhesion mechanisms. This differentialeffect may offer an opportunity for therapeutic intervention.

In summary, our studies of CTCs in both breast and prostate cancerpatients and mouse models point to CTC clusters as critical mediators ofcancer metastasis. These coexist with single migratory CTCs, making acontribution to the metastatic burden that far exceeds theircomparatively small numbers in the circulation. The ability of tumorcell aggregates to detach from a primary tumor and maintain theircohesion as they survive in the bloodstream may identify a novel andpotentially targetable step in the bloodborne dissemination of cancer.

Experimental Procedures

CTC Capture and Identification

Blood specimens for CTC analysis were obtained after informed patientconsent, per institutional review board (IRB) protocol (05-300), at theMassachusetts General Hospital. A maximum of 20 ml of blood was drawn inEDTA vacutainers. Within 4 hr from blood draw, 3 ml of blood wasprocessed through the HBCTC-Chip or 6-12 ml of blood was processedthrough the negCTC-iChip.

For mouse studies, blood was retrieved via cardiac puncture and 1 ml ofblood was processed through the HBCTC-Chip.

HBCTC-Chips were manufactured on site at the Massachusetts GeneralHospital Cancer Center/BioMEMS Resource Facility. For patient samplesand mouse xenografts, chips were functionalized as previously described(Yu et al., 2013) with a cocktail of 10 mg/ml each of biotinylatedantibodies against EpCAM (R&D Systems), EGFR (Cetuximab, Lilly), andHER2 (R&D Systems). For 4T1 mouse mammary tumor cells, chips werefunctionalized with a cocktail of antibodies against mouse EpCAM(BioLegend) and EGFR (Cetuxi-mab, Lilly). Samples from patients withprostate cancer were processed as described (Miyamoto et al., 2012).negCTC-iChips were designed and fabricated as previously described(Ozkumur et al., 2013).

Tumorigenesis Assays

All mouse experiments were carried out in compliance with institutionalguidelines. For tail vein experiments, NOD SCID Gamma (NSG) mice(Jackson Labs) were injected with 2×105 LM2 cells, 4×105 BT474 cells, or2×105 4T1 cells and monitored with IVIS Lumina II (CaliperLifeSciences). For CTC clusters metastatic potential assessment, 2×106LM2-GFP (or 4T1-GFP) and 2×106 LM2-mCherry (or 4T1-mCherry) cells wereprepared separately or mixed 1:1, suspended in 100 ml of 50% BasementMembrane Matix Phenol Red-free (BD Biosciences) in PBS and injectedorthotopically in NSG mice. Blood draw for CTCs enumeration wasperformed 4 weeks after tumor onset. For pla-koglobin knockdownexperiments, 1×106 LM2-CTRL or LM2-Plakoglobin shRNA cells weresuspended in 100 ml of 50% Basement Membrane Matrix Phenol Red-free inPBS and injected orthotopically in NSG mice. Blood draw for CTCsenumeration and lung metastasis analysis were performed 4 weeks aftertumor onset.

Analysis of RNA Sequencing Data

Determination of reads-per-million (rpm): color space reads were alignedusing tophat and bowtiel with the no-novel-juncs argument set with humangenome version hg19 and transcriptome defined by the hg19 knownGenetable from genome.ucsc.edu. Reads that did not align or aligned tomultiple locations in the genome were discarded. The hg19 tableknownToLocusLink from genome.ucsc.edu was used to map, if possible, eachaligned read to the gene whose exons the read had aligned to. The readscount for each gene was the number of reads that were so mapped to thatgene. This count was divided by the total number of reads that weremapped to any gene and multiplied by one million to form thereads-per-million (rpm) count. We used rpm rather than rpkm because wenoted a 30 bias in the alignments.

Accession Numbers

The Gene Expression Omnibus accession number for the sequencing datareported in this paper is GSE51827.

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Extended Experimental Procedures

CTC Capture and Identification

Cells captured on the HBCTC-Chip were fixed with 4% paraformaldehyde andwashed with PBS. Fixed cells were then permeabilized with 1% NP40 inPBS, blocked with 3% goat serum/2% BSA, and immunostained withantibodies against wide spectrum cytokeratin (Abcam), prostate specificantigen (DAKO), prostate-specific membrane antigen (obtained from N.Bander), CD45 (Abcam), plako-globin (Sigma Aldrich) and DAPI.Alternatively, GFP- or mCherry-expressing cells captured on chip werewashed with PBS and imaged directly. Stain-positive cells were detectedusing the BioView™ Ltd. automated imaging system (Billerica, Mass.).High-resolution images were obtained with an upright fluorescencemicroscope (Eclipse 90i, Nikon, Melville, N.Y.).

negCTC-iChips were designed and fabricated as previously described(Ozkumur et al., 2013). Before processing, whole blood samples wereexposed to biotinylated antibodies against CD45 (R&D Systems) and CD66b(AbD Serotec, biotinylated in house) and then incubated with DynabeadsMyOne™ Streptavidin T1 (Invitrogen) to achieve magnetic labeling anddepletion of white blood cells (Oz-kumur et al., 2013). The CTC-enrichedproduct was stained in solution with Alexa488-conjugated antibodiesagainst EpCAM (Cell Signaling Technology), Cadherin 11 (R&D Systems) andHER2 (Biolegend) to identify CTCs, and TexasRed-conjugated antibodiesagainst CD45 (BD Biosciences), CD14 (BD Biosciences) and CD16 (BDBiosciences) to identify contaminating white blood cells.

Assessment of Metastasis-free Survival and Overall Survival

Kaplan-Meier survival curves based on clinical data from patients atMassachusetts General Hospital were generated with XLStat™ software(Addinsoft). For “plakoglobin high” versus “plakoglobin low” distantmetastasis-free survival in breast cancer patients (as well as for theother CTC-clusters-associated genes) we identified publically availablehuman primary breast cancer gene expression data sets and samples withinthem having the following characteristics: a) distant-metastasis-freesurvival information was available, b) there was no evidence ofneo-adjuvant treatment, c) the platform used to measure gene expressionmeasured at least 10,000 transcripts, d) if there were multiple samplesfor a patient, only one was used, e) there were at least 40 samples inthe data set satisfying the preceding criteria. The following data setswere used (Bos et al., 2009; Chanfion et al., 2008; Chin et al., 2006;Desmedt et al., 2007; Li et al., 2010; Loi et al., 2008; Ma et al.,2004; Minn et al., 2005, 2007; Schmidt et al., 2008; Sotiriou et al.,2006; van 't Veer et al., 2002; van de Vijver et al., 2002; Wang et al.,2005). For each data set, we identified all probes or probesets forplakoglobin and used the one with greatest standard deviation across thesamples of the data set. For each data set we characterized a sample as“high plakoglobin” if its plakoglobin expression was in the top third ofplakoglobin expression for that data set and as “low plakoglobin”otherwise. We then created a Kaplan-Meier plot and calculated a logranktwo-sided p value using the distant-metastasis-free survival informationfor the samples from all the data sets and the “high plakoglobin” versus“low plakoglobin” classification.

Single-Cell Micromanipulation

The CTC-enriched product was collected in a 35 mm petri dish and viewedusing a Nikon Eclipse™ Ti inverted fluorescent microscope. Single CTCsand CTC clusters were identified based on intact cellular morphology,Alexa488-positive staining and lack of TexasRed staining. Target cellswere individually micromanipulated with a 10 mm transfer tip on anEppendorf TransferMan™ NK 2 micromanip-ulator and ejected into PCR tubescontaining RNA protective lysis buffer (10×PCR Buffer II, 25 mM MgCl2,10% NP40, 0.1 M DTT, SUPERase-In, Rnase Inhibitor, 0.5 uM UP1 Primer, 10mM dNTP and Nuclease-free water) and immediately flash frozen in liquidnitrogen.

Single-Cell RNA Amplification and Sequencing

RNA samples extracted from CTCs were thawed on ice and incubated at 70°C. for 90 s. To generate cDNA, samples were treated with reversetranscription master mix (0.05 uL RNase inhibitor, 0.07 uL T4 gene 32protein, and 0.33 uL SuperScript III™ Reverse Transcrip-tase per 1×volume) and incubated on thermocycler at 50° C. for 30 min and 70° C.for 15 min. To remove free primers, 1.0 uL of EXOSAP™ mix was added toeach sample, which was incubated at 37° C. for 30 min and inactivated at80° C. for 25 min. Next, a 3′-poly-A tail was added to the cDNA in eachsample by incubating in master mix (0.6 uL 10×PCR Buffer II, 0.36 uL 25mM MgCl2, 0.18 uL 100 mM dATP, 0.3 uL Terminal Transferase, 0.3 uL RNaseH, and 4.26 uL H₂O per 1× volume) at 37° C. for 15 min and inactivatedat 70° C. for 10 min. A second strand cDNA was synthesized by dividingeach sample into 4 and incubating in master mix (2.2 uL 10× HighFidelity PCR Buffer, 1.76 uL 2.5 mM each dNTP, 0.066 uL UP2 Primer at100 uM, 0.88 uL 50 mM MgSO4, 0.44 uL Platinum Taq DNA Polymerase, and13.654 uL H₂O per 1× volume) at 95° C. for 3 min, 50° C. for 2 min, and72° C. for 10 min. PCR amplification (95° C. for 3 min, 20 cycles of 95°C. for 30 s, 67° C. for 1 min, and 72° C. for 6 min 6 s) was performedwith master mix (4.1 uL 10× High Fidelity PCR Buffer, 1.64 uL 50 mMMgSO4, 4.1 uL 2.5 mM each dNTP, 0.82 uL AUP1 Primer at 100 uM, 0.82 uLAUP2 Primer at 100 uM, 0.82 uL Platinum Taq DNA Polymerase, and 6.7 uLH₂O per 1× volume). The 4 reactions of each sample were pooled andpurified using the QIAGEN PCR Purification Kit (Cat. No 28106) andeluted in 50 uL EB buffer. Samples were selected by testing for genesGapdh, ActB, Ptprc (CD45), Krt8, Krt18 and Krt19 using qPCR. Each samplewas again divided in 4 and a second round of PCR amplification (9 cyclesof 98° C. for 3 min, 67° C. for 1 min, and 72° C. for 6 min 6 s) wasperformed with master mix (9 uL 10× High Fidelity PCR Buffer, 3.6 uL 50mM MgSO4, 13.5 uL 2.5 mM each dNTP, 0.9 uL AUP1 Primer at 100 uM, 0.9 uLAUP2 Primer at 100 uM, 1.8 uL Platinum Taq DNA Polymerase, and 59.1 uLH₂O per 1× volume). Samples were pooled and purified using AgencourtAMPure XP beads and eluted in 40 uL 1× low TE buffer.

Sequencing Library Construction

To shear the DNA using the Covaris S2™ System, 1× low TE buffer and 1.2uL shear buffer were added to each sample. Conditions of the shearingprogram include: 6 cycles, 5° C. bath temperature, 15° C. bathtemperature limit, 10% duty cycle, intensity of 5, 100 cy-cles/burst,and 60 s. Then, samples were end-polished at room temperature for 30 minwith a master mix (40 uL 5× Reaction Buffer, 8 uL 10 mM dNTP, 8 uL EndPolish Enzymel, 10 uL End Polish Enzyme2, and 14 uL H₂O per 1× volume).DNA fragments larger than 500 bp were removed with 0.5× volumes ofAgencourt AMPure XP™ beads. Supernatant was transferred to separatetubes. To size-select 200-500 bp DNA products, 0.3× volumes of beadswere added and samples were washed twice with 70% EtOH. The productswere eluted in 36 uL low TE buffer. A dA-tail was added to eachsize-selected DNA by treating with master mix (10 uL 5× Reaction Buffer,luL 10 mM dATP, and 5 uL A-Tailing Enzyme I per 1× volume) and incubatedat 68° C. for 30 min and cooled to room temperature. To label anddistinguish each DNA sample for sequencing, barcode adaptors (5500 SOLiD4464405) were ligated to DNA using the 5500 SOLiD Fragment LibraryEnzyme Module (4464413). Following barcoding, samples were purifiedtwice using the Agencourt AMPure XP™ beads and eluted in 22 uL low TEbuffer. Following a round of PCR Amplification (95° C. for 5 min, 12cycles of 95° C. for 15 s, 62° C. for 15 s, and 70° C. for 1 min, and70° C. for 5 min), the libraries were purified with AMPure XP beads.Finally, to quantify the amount of ligated DNA, SOLiD Library TaqMan™Quantitation Kit was used to perform qPCR. Completed barcoded librarieswere then subjected to emulsion PCR with template beads preparation andsequenced on the ABI 5500XL™

Analysis of RNA Sequencing Data

Clustering: the minimum of 1 and the smallest positive value of the rpmmatrix was added to the rpm matrix to eliminate zeros. The result wasthen log transformed and median polished. The rows (corresponding togenes) with the top 2000 standard deviations were retained and the restof the rows discarded. The result was clustered using agglomerativehierarchical clustering with average linkage with distance metric equalto 1 minus the Pearson correlation coefficient.

Supervised differential gene expression: samples that showed highexpression of contaminant WBC markers and no expression of CTC markersat the RNA level were excluded from the analysis. For each pair ofsingle CTCs sample and CTC cluster sample from the same patient, wecalculated a FDR q-value and a normalized fold change using the DEGexpfunction of version 1.10.0 of the Bio-conductor DEGseg™ package (Wang etal., 2010) with method set to ‘MARS’ and q-values calculated usingBenjamini-Hochberg. For each pair and direction (e.g., up in CTCclusters versus single CTCs) a gene was considered a hit if its q-valuewas less than 0.01 and its fold change was greater than 2. Then, foreach direction, we considered the genes that were hits for 70% or moreof the pairs. The desmosome (resp. adherence junction) metagene wasdefined to be the mean over the desmosome (resp. adherence junction)marker genes of the normalized log 2 fold change between the CTCclusters and the single CTCs as determined by DEGseg™

In Vivo Flow Cytometry

DiD-labeled LM2 single or clustered cells were adoptively transferredintravenously and detected in the peripheral circulation (Novak et al.,2004). Of note, single and clustered LM2 cells were injected separatelyin different animals to avoid signal misinterpretation. DiD was excitedby a 635 nm laser and detected with a 695±27.5 nm bandpass filter usinga photomultiplier tube. Circulation kinetics for LM2-SCs and LM2-CLswere quantified using MATLAB™ (Mathworks).

Immunohistochemistry

Formalin-fixed and paraffin embedded mouse xenografts primary tumors,lung metastases, as well as human primary tumors and matched metastaticlesions were sectioned and stained overnight at 4° C. with antibodiesagainst cleaved caspase 3 (Cell Signaling Technology), GFP (Cellsignaling Technology), mCherry (Abeam), plakoglobin (Sigma Aldrich) andCD31 (Abcam). GFP/mCherry and Plakoglobin/CD31 double-stainings wereperformed with EnVision G/2 Doublestain System (Dako). All specimenswere counter-stained with Hematoxylin. Images of the whole tissue weretaken with ScanScope™ (Aperio).

Cell Culture and Reagents

HMEC, MCF10A, BT474, MCF7, T47D, 4T1, BT549, BT20 and ZR-75-1 cells werepurchased from the American Type Culture Collection (ATCC) andpropagated according to the manufacturer's instructions. MDA-MB-231 LM2cells were propagated in DMEM (Life Technologies) supplemented with 10%fetal bovine serum (Life Technologies). To generate BT474, 4T1 or LM2single cells or clusters for tail vein injections, cells growing inmonolayer at 80% confluence were incubated with trypsin (LifeTechnologies) for one minute to generate floating clusters. Clusterswere then distributed equally in two separate dishes. In one of the twodishes, clusters were mechanically dissociated by pipetting to generatea single-cell suspension.

Cell-to-cell adhesion assay was performed with the Vybrant™ Cell-to-CellAdhesion Assay Kit (Invitrogen) according to the manu-facturer'sinstructions.

The plasmid expressing mCherry was purchased from Addgene. PlakoglobinTRC shRNAs were purchased from Thermo Scientific. Lentiviral packagingvectors (Addgene) were used to transfect 293T cells (ATCC) and producelentiviral particles. Infections of target cells lines was performedovernight at a MOI=10 in growth medium containing 8 mg/ml polybrene(Thermo Scientific).

SUPPLEMENTAL REFERENCES

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What is claimed herein is:
 1. A method of treating breast or epithelialcancer, the method comprising administering a treatment to prevent orreduce metastasis in a subject determined to have a level of CTC clusterwhich is increased relative to a control level.
 2. The method of claim1, the method further comprising not administering a treatment toprevent or reduce metastasis in a subject determined to have a level ofCTC clusters is not increased relative to a control level.
 3. The methodof claim 1, wherein the treatment to prevent or reduce metastasis isselected from the group consisting of: an inhibitor of a CTC-C markergene selected from the list of Table 2, 3, or 4; chemotherapy; radiationtherapy; or removal of a tumor.
 4. The method of claim 1, wherein notadministering a treatment can comprise a clinical approach of monitoringwithout therapeutic intervention.
 5. The method of claim 1, wherein thelevel of CTC clusters is measured by measuring the expression level of aCTC cluster (CTC-C) marker gene in the sample obtained from the subject;wherein the CTC-C marker gene is a gene selected from the list of Table2, 3, or
 4. 6. The method of claim 5, wherein the CTC-C marker gene isplakoglobin.
 7. The method of claim 5, wherein the expression level of aCTC-C marker gene in circulating tumor cells in the sample is measured.8. The method of claim 5, wherein the expression level of a CTC-C markergene in cancer cells obtained from the subject is measured.
 9. Themethod of claim 1, wherein the level of CTC clusters is measured using a^(HB)CTC-Chip.
 10. The method of claim 1, wherein the subject is asubject in need of treatment for cancer.
 11. The method of claim 1,wherein an increased level of CTC clusters is a level at least 1.5×greater than the control level.
 12. The method of claim 6, wherein anincreased level of plakoglobin expression is a level at least 1.5×greater than the control level.
 13. The method of claim 1, furthercomprising a first step of measuring the level of circulating tumor cell(CTC) clusters in a sample obtained from a subject with a breast orepithelial cancer.
 14. A method of treating cancer metastasis, themethod comprising reducing the level of expression or activity of aCTC-C marker gene; wherein the CTC-C marker gene is a gene selected fromthe list of Table 2, 3, or
 4. 15. The method of claim 14, whereinreducing the level of expression or activity of a CTC-C marker genecomprises administering a CTC-C marker gene inhibitory nucleic acid. 16.The method of claim 15, wherein the inhibitory nucleic acid is a siRNA.17. The method of claim 14, wherein the CTC-C marker gene isplakoglobin.
 18. A method of reducing the level of circulating tumorcell (CTC) clusters in a subject with cancer, the method comprisingreducing the level of expression or activity of a CTC-C marker gene;wherein the CTC-C marker gene is a gene selected from the list of Table2, 3, or 4.